Methods and Systems for Detecting Prostaglandins by LC-MS/MS

ABSTRACT

Disclosed are methods, systems, and computer program products for using liquid chromatography/tandem mass spectrometry (LC-MS/MS) for the analysis of endogenous biomarkers, such as PGD 2 , in a biological sample. More specifically, the methods, systems, and computer program products are described for detecting and quantifying the amount of an PGD 2  in a sample. The quantitative analysis may be helpful in making clinical diagnoses.

RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applicationNo. 63/076,544 filed on Sep. 10, 2020. The disclosure of U.S.provisional patent application No. 63/076,544, is incorporated byreference in its entirety herein.

FIELD OF THE INVENTION

The presently disclosed subject matter relates to methods and systemsfor the analysis of the prostaglandins such as prostaglandin D₂ (PGD₂).In certain embodiments, the measurement of PGD₂ may be used for clinicaldiagnosis.

BACKGROUND

Prostaglandins are fatty acids derived from arachidonic acid metabolism.One such prostaglandin, prostaglandin D₂ (PGD₂), is derived mainly fromprostaglandin H2. The activity of PDG₂ is highest in the brain, spinalcord, intestines, and stomach, and PGD₂ is the major prostaglandinproduced by uterine tissue. PGD₂ is involved in a variety ofphysiological functions, including regulation of body temperature,hormone release, modulation of pain response, and sleep-wake cycles.PGD₂ is a known bronchoconstrictor, neuromodulator, andanti-antithrombin agent. PGD₂ has also been demonstrated to have ananti-metastatic effect on malignant tumor cells. Thus, PGD₂ has beendemonstrated to play a role in both inflammatory and homeostaticfunctions.

PGD₂ a known biomarker of several clinical conditions. For example, PGD₂is a major eicosanoid product of mast cells and is released in largequantities during allergic and asthmatic analaphylaxis (Roberts, L. J.,II, and Sweetman, B. J., “Metabolic fate of endogenously synthesizedprostaglandin D₂ in a human female with mastocytosis,” Prostaglandins30(3), 383-400 (1985)). Mastocytosis patients produce excessive amountsof PGD₂, which causes vasodilation, flushing, hypotension, and syncopalepisodes (Roberts et al., 1985). PGD₂ is also produced in the brain viaan alternative pathway involving a soluble, secreted PGD-synthase alsoknown as β-trace (Hayaishi, O., “Sleep-wake regulation by prostaglandinsD₂ and E₂ ,” J. Biol. Chem. 263(29), 14593-14596 (1988) and Onoe, H., etal., “Prostaglandin D₂, a cerebral sleep-inducing substance inmonkeys.,” Proc. Natl. Acad. Sci. U.S.A. 85(11), 4082-4086 (1988)). Inthe brain, PGD₂ produces normal physiological sleep and lowering of bodytemperature (Hayaishi, 1988 and Onoe et al., 1988). Furtherpharmacological actions include inhibition of platelet aggregation andrelaxation of vascular smooth muscle (Giles, H., and Leff, P., “Thebiology and pharmacology of PGD₂ ” Prostaglandins 35(2), 277-300(1988)). PGD₂ inhibits human ovarian tumor cell proliferation with anIC₅₀ of 6.8 μM (Kikuchi, Y. et al., “Preclinical studies of antitumorprostaglandins by using human ovarian cancer cells,” Cancer MetastasisRev. 13(3-4), 309-315 (1994)).

Thus, there is a need to develop analytical techniques that can be usedfor the measurement of prostaglandins such as PGD₂.

SUMMARY

Embodiments of the present disclosure comprise compositions, methods,and systems for the detection of prostaglandins. In an embodiment, theprostaglandin is PGD₂. The present disclosure may be embodied in avariety of ways.

In certain embodiments, disclosed is a method for determining thepresence or amount of at least one biomarker of interest in a sample,the method comprising: providing a sample believed to contain at leastone biomarker of interest; chromatographically separating the at leastone biomarker of interest from other components in the sample; andanalyzing the chromatographically separated at least one biomarker ofinterest by mass spectrometry to determine the presence or amount of theat least one biomarker of interest in the sample.

In some embodiments, the biomarker of interest is a prostaglandin. Inone embodiment, the prostaglandin is PGD₂. Or, the biomarker of interestmay be other prostaglandins. In some embodiments, the disclosed subjectmatter provides methods and systems for the quantitative analysis ofPGD₂. In an embodiment, the methods and systems of the disclosure allowfor measurement of PGD₂ without the need for derivatization processes.

For example, in one embodiment, disclosed is a method for determiningthe presence or amount of PGD₂ in a sample by tandem mass spectrometry.The method may comprise any one of the steps of: (a) obtaining a samplefrom a subject; (b) optionally adding a stable isotope labeled PGD₂ tothe sample as an internal standard; (c) performing HPLC; and (d)measuring the PGD₂ (both labeled and unlabeled) by tandem massspectrometry. In an embodiment, the tandem mass spectrometry maycomprise the steps of: (i) generating a precursor ion of the PGD₂; (ii)generating one or more fragment ions of the precursor ion; and (iii)detecting the presence or amount of the precursor ion generated in step(i) and/or the at least one or more fragment ions generated in step(ii), or both, and relating the detected ions to the presence or amountof the PGD₂ in the sample. In an embodiment, the step of relating thedetected ions to the presence or amount of the PGD₂ in the sample isquantitative. In an embodiment, the tandem mass spectrometry is coupledto HPLC. The HPLC step may directly precede the tandem mass spectrometryanalysis (i.e., LC-MS/MS). In some embodiments, the HPLC is highturbulence liquid chromatography (HTLC). In some embodiments, solidphase extraction is used to partially purify the PGD₂ prior to HPLC.Also in some embodiments, a second stable isotope labeled PGD₂ (i.e., adifferent isotope) is added to the sample as an internal standard afterthe extraction but prior to the LC-MS/MS. In some embodiments, duplicatesets of charcoal stripped calibrators are analyzed in each batch. Themethod may alternatively be used to measure other prostaglandins, e.g.,PGI₂, PGE₂ or PGF and subtypes thereof.

Another aspect of the disclosure is a system for performing the methods.In some embodiments, the system comprises: a station or component forproviding a test sample suspected of containing PGD₂; a station orcomponent for partially purifying the PGD₂ from other components in thesample; a station or component for chromatographically separating PGD₂from other components in the sample; and a station or component foranalyzing the chromatographically separated PGD₂ by mass spectrometry todetermine the presence or amount of the PGD₂ in the test sample. Thesystem may alternatively be used to measure other prostaglandins, e.g.,PGI₂, PGE₂ or PGF and subtypes thereof.

An additional aspect of the disclosure is a computer program producttangibly embodied in a non-transitory machine-readable storage medium,including instructions configured to cause one or more data processorsto perform any of the method steps or to control any of the stations orcomponents of the system. For example, the computer program product maycontain instructions to perform actions to measure the presence oramount of PGD₂ in a sample comprising at least one of the followingsteps: (a) obtaining a sample from a subject; (b) optionally adding astable isotope-labeled PGD₂ to the sample as an internal standard; (c)performing solid phase extraction; (d) performing HTLC; and (e)measuring the PGD₂ by tandem mass spectrometry.

Certain objects of the disclosure, having been stated hereinabove, willbecome further evident as the description proceeds when taken inconnection with the accompanying figures and examples as describedherein below.

BRIEF DESCRIPTION OF THE FIGURES

The present disclosure may be better understood by referring to thefollowing non-limiting figures.

FIG. 1 shows a flow chart of a method for quantitative analysis of PGD₂in accordance with one embodiment of the present disclosure.

FIG. 2 shows a system for quantitative analysis of PGD₂ in accordancewith one embodiment of the present disclosure.

FIG. 3 shows an exemplary computing device in accordance with variousembodiments of the disclosure.

FIG. 4 shows a diagram for an on-line LC-MS/MS system in accordance withone embodiment of the present disclosure.

FIG. 5 depicts a plot of a PGD₂ system suitability testing (SST)chromatogram in accordance with an embodiments of the presentdisclosure.

DETAILED DESCRIPTION OF THE INVENTION

The ensuing description provides preferred exemplary embodiments only,and is not intended to limit the scope, applicability or configurationof the disclosure. Rather, the ensuing description of the preferredexemplary embodiments will provide those skilled in the art with anenabling description for implementing various embodiments. It isunderstood that various changes may be made in the function andarrangement of elements without departing from the spirit and scope asset forth in the appended claims.

Specific details are given in the following description to provide athorough understanding of the embodiments. However, it will beunderstood that the embodiments may be practiced without these specificdetails. For example, circuits, systems, networks, processes, and othercomponents may be shown as components in block diagram form in order notto obscure the embodiments in unnecessary detail. In other instances,well-known circuits, processes, algorithms, structures, and techniquesmay be shown without unnecessary detail in order to avoid obscuring theembodiments.

Abbreviations

-   -   AS=auto sampler    -   APCI=atmospheric pressure chemical ionization    -   DB=double blank    -   ESI=electrospray ionization    -   HTLC=high turbulence (throughput) liquid chromatography    -   HPLC=high performance liquid chromatography    -   IS=internal standard    -   LC-MS/MS=liquid chromatography with tandem mass spectrometry    -   LLE=liquid-liquid extraction    -   LOQ=limits of quantification    -   LLOQ=lower limit of quantification    -   SRM—selected reaction monitoring    -   SST=system suitability test    -   ULOQ=upper limit of quantification

Definitions

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those well-known and commonly used in the art. Known methodsand techniques are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are discussed herein unless otherwiseindicated. Enzymatic reactions and purification techniques are performedaccording to manufacturer's specifications, as commonly accomplished inthe art or as described herein. The nomenclatures used in connectionwith the laboratory procedures and techniques described herein are thosewell-known and commonly used in the art.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Moreover, all ranges disclosed hereinare to be understood to encompass any and all subranges subsumedtherein. For example, a stated range of “1 to 10” should be consideredto include any and all subranges between (and inclusive of) the minimumvalue of 1 and the maximum value of 10; that is, all subranges beginningwith a minimum value of 1 or more, e.g. 1 to 6.1, and ending with amaximum value of 10 or less, e.g., 5.5 to 10. Additionally, anyreference referred to as being “incorporated herein” is to be understoodas being incorporated in its entirety.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

As used herein, the terms “a”, “an”, and “the” can refer to one or moreunless specifically noted otherwise.

The use of the term “or” is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive, although the disclosure supports a definition that refers toonly alternatives and “and/or.” As used herein “another” can mean atleast a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among samples.

The term “biological sample” encompasses any sample obtained from abiological source. A biological sample can, by way of non-limitingexample, include blood, plasma, amniotic fluid, sera, urine, feces,epidermal sample, skin sample, cheek swab, sperm, cultured cells, bonemarrow sample and/or chorionic villi. Convenient biological samples maybe obtained by, for example, scraping cells from the surface of thebuccal cavity. The term biological sample encompasses samples which havebeen processed to release or otherwise make available a prostaglandinfor detection as described herein. The biological sample may be obtainedfrom a stage of life such as a fetus, young adult, adult, and the like.Fixed or frozen tissues also may be used. In some embodiments, the testsample is not a biological sample, but comprises a non-biologicalsample, e.g., obtained during the manufacture or laboratory analysis ofa synthetic analyte, which can be analyzed to determine the compositionand/or yield of the manufacturing and/or analysis process.

As used herein, the terms “subject,” “individual,” and “patient” areused interchangeably. The use of these terms does not imply any kind ofrelationship to a medical professional, such as a physician. A “subject”may be an animal. Thus, in some embodiments, the biological sample isobtained from a mammalian animal, including, but not limited to a human,a dog, a cat, a horse, a rat, a monkey, and the like. In someembodiments, the biological sample is obtained from a human subject. Insome embodiments, the subject is a patient, that is, a living personpresenting themselves in a clinical setting for diagnosis, prognosis, ortreatment of a disease or condition.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the phrase “liquid chromatography” or “LC” is used torefer to a process for the separation of one or more molecules oranalytes in a sample from other analytes in the sample. LC involves theslowing of one or more analytes of a fluid solution as the fluiduniformly moves through a column of a finely divided substance. Theslowing results from the distribution of the components of the mixturebetween one or more stationery phases and the mobile phase. LC includes,for example, reverse phase liquid chromatography (RPLC) and highpressure liquid chromatography (HPLC). In some cases, LC refers toreverse phase LC with a hydrophobic stationary phase in combination witha mobile phase comprised of water and/or water-miscible organicsolvents, such as methanol or acetonitrile. In some case, LC may referto ion exchange chromatography, affinity chromatography, normal phaseliquid chromatography, or hydrophilic interaction chromatography.

As used herein, the term “HPLC” or “high performance liquidchromatography” refers to liquid chromatography in which the degree ofseparation is increased by forcing the mobile phase under pressurethrough a stationary phase, typically a densely packed column. Thechromatographic column typically includes a medium (i.e., a packingmaterial) to facilitate separation of chemical moieties (i.e.,fractionation). The medium may include minute particles. The particlescan include a bonded surface that interacts with the various chemicalmoieties to facilitate separation of the chemical moieties such as thebiomarker analytes quantified in the experiments herein. One suitablebonded surface is a hydrophobic bonded surface such as an alkyl bondedsurface. Alkyl bonded surfaces may include C-4, C-8, or C-18 bondedalkyl groups, preferably C-18 bonded groups. The chromatographic columnincludes an inlet port for receiving a sample and an outlet port fordischarging an effluent that includes the fractionated sample. In themethod, the sample (or pre-purified sample) may be applied to the columnat the inlet port, eluted with a solvent or solvent mixture, anddischarged at the outlet port. Different solvent modes may be selectedfor eluting different analytes of interest. For example, liquidchromatography may be performed using a gradient mode, an isocraticmode, or a polytyptic (i.e. mixed) mode.

As used herein, the term “HTLC” refers to high turbulence liquidchromatography Liquid chromatography may, in certain embodiments,comprise high turbulence liquid chromatography or high throughput liquidchromatography (HTLC). See, e.g., Zimmer et al., J. Chromatogr. A854:23-35 (1999); see also, U.S. Pat. Nos. 5,968,367; 5,919,368;5,795,469; and 5,772,874. Traditional HPLC analysis relies on columnpackings in which laminar flow of the sample through the column is thebasis for separation of the analyte of interest from the sample. In suchcolumns, separation is a diffusional process. Turbulent flow, such asthat provided by HTLC columns and methods, may enhance the rate of masstransfer, improving the separation characteristics provided.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof the components of a test sample matrix. Preferably, the componentseluted from the analytical column are separated in such a way to allowthe presence or amount of an analyte(s) of interest to be determined. Insome embodiments, the analytical column comprises particles having anaverage diameter of about 3-10 μm. Or, for HTLC the analytical columnmay comprise particles having an average diameter of about 25-75 μm. Insome embodiments, the analytical column is a functionalized silica orpolymer-silica hybrid, or a polymeric particle or monolithic silicastationary phase, such as a phenyl-hexyl functionalized analyticalcolumn.

Analytical columns can be distinguished from “extraction columns,” whichtypically are used to separate or extract retained materials fromnon-retained materials to obtained a “purified” sample for furtherpurification or analysis. In some embodiments, the extraction column isa functionalized silica or polymer-silica hybrid or polymeric particleor monolithic silica stationary phase, such as a Poroshell SBC-18column.

As used herein the term “capillary electrophoresis” (CE) refers to aprocess for the separation of one or more molecules or analytes in asample from other analytes in the sample, based on their ionic mobilityin an electrolyte solution while exposed to an electric field. CEincludes, for example, capillary zone electrophoresis (CZE).

As used herein, the term “separate” or “purify” or the like are not usednecessarily to refer to the removal of all materials other than theanalyte of interest from a sample matrix. Instead, in some embodiments,the terms are used to refer to a procedure that enriches the amount ofone or more analytes of interest relative to one or more othercomponents present in the sample matrix. In some embodiments, a“separation” or “purification” may be used to remove or decrease theamount of one or more components from a sample that could interfere withthe detection of the analyte, for example, by mass spectrometry.

As used herein, the term “mass spectrometry” or “MS” refers to atechnique for the identification and/or quantitation of molecules in asample. MS includes ionizing the molecules in a sample to form chargedmolecules (ions) in gas phase; separating the charged moleculesaccording to their mass-to-charge (m/z) ratio; and detecting the chargedmolecules. MS allows for both the qualitative and quantitative detectionof molecules in a sample. The molecules may be ionized and detected byany suitable means known to one of skill in the art. As used herein, a“mass spectrometer” is an apparatus that includes a means for ionizingmolecules and detecting charged molecules.

In certain embodiments, “tandem mass spectrometry” (MS/MS) is used.Tandem mass spectrometry (MS/MS) is the name given to a group of massspectrometric methods wherein “parent or precursor” ions generated froma sample are fragmented to yield one or more “fragment, daughter orproduct” ions, which are subsequently mass analyzed by a second MSprocedure. As used herein, parent and precursor ion are usedinterchangeably. Also, as used herein fragment and product ions are usedinterchangeably. As used herein, fragment, daughter and product ions areused interchangeably. MS/MS methods are useful for the analysis ofcomplex mixtures, especially biological samples, in part because theselectivity of MS/MS can minimize the need for extensive sample clean-upprior to analysis. In an example of an MS/MS method (i.e., triplequadrupole MS/MS), precursor ions are generated from a sample and passedthrough a first mass filter (quadrupole 1 or Q1) to select those ionshaving a particular mass-to-charge ratio. These ions are thenfragmented, typically by collisions with neutral gas molecules in thesecond quadrupole (Q2), to yield product (fragment) ions which areselected in the third quadrupole (Q3), the mass spectrum of which isrecorded by an electron multiplier detector. The product ion spectra soproduced are indicative of the structure of the precursor ion, and thetwo stages of mass filtering can eliminate ions from interfering speciespresent in the conventional mass spectrum of a complex mixture.

The term “ionization” and “ionizing” as used herein refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those ions havinga net negative charge of one or more electron units, while positive ionsare those ions having a net positive charge of one or more electronunits.

The term “electron ionization” as used herein refers to methods in whichan analyte of interest in a gaseous or vapor phase interacts with a flowof electrons. Impact of the electrons with the analyte produces analyteions, which may then be subjected to a mass spectrometry technique.

The term “chemical ionization” as used herein refers to methods in whicha reagent gas (e.g. ammonia) is subjected to electron impact, andanalyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

The term “field desorption” as used herein refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

The term “desorption” as used herein refers to the removal of an analytefrom a surface and/or the entry of an analyte into a gaseous phase.

The term “matrix-assisted laser desorption ionization,” or “MALDI” asused herein refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photo-ionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

The term “surface enhanced laser desorption ionization,” or “SELDI” asused herein refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photo-ionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

The term “electrospray ionization,” or “ESI,” as used herein refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Upon reaching the end of the tube, the solution maybe vaporized (nebulized) into a jet or spray of very small droplets ofsolution in solvent vapor. This mist of droplet can flow through anevaporation chamber which is heated slightly to prevent condensation andto evaporate solvent. As the droplets get smaller the electrical surfacecharge density increases until such time that the natural repulsionbetween like charges causes ions as well as neutral molecules to bereleased.

The term “Atmospheric Pressure Chemical Ionization,” or “APCI,” as usedherein refers to mass spectroscopy methods that are similar to ESI,however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then, ions are typically extracted into a mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “Atmospheric Pressure Photoionization” (“APPI”) as used hereinrefers to the form of mass spectroscopy where the mechanism for thephotoionization of molecule M is photon absorption and electron ejectionto form the molecular M+. Because the photon energy typically is justabove the ionization potential, the molecular ion is less susceptible todissociation. In many cases it may be possible to analyze sampleswithout the need for chromatography, thus saving significant time andexpense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant.

The term “inductively coupled plasma” as used herein refers to methodsin which a sample is interacted with a partially ionized gas at asufficiently high temperature to atomize and ionize most elements.

As used herein, the term “isotopically labeled,” “stable isotopicallylabeled” or “stable isotope labeled” or similar such terms encompassesthe process or product, respectively, of enriching a molecule with anon-radioactive isotope of a given atom so as to alter the average massof said atom within a molecule and thereby alter the average mass ofsaid molecule. Generally, this is accomplished by replacing the lightisotopes more frequently found in nature and in natural molecules (e.g.,carbon-12 or nitrogen-14), with the less common heavy isotopes (e.g.,carbon-13 or nitrogen-15).

As used herein, a “quadrupole analyzer” is a type of mass analyzer usedin MS. It consists of four circular rods (two pairs) that are set highlyparallel to each other. The quadrupole may be in triple quadrupoleformat as is known in the art. The quadrupole analyzer is the componentof the instrument that organizes the charged particles of the samplebased on their mass-to-charge ratio. One of skill in the art wouldunderstand that use of a quadrupole analyzer can lead to increasedspecificity of results. One pair of rods is set at a positive electricalpotential and the other set of rods is at a negative potential. To bedetected, an ion must pass through the center of a trajectory pathbordered and parallel to the aligned rods. When the quadrupoles areoperated at a given amplitude of direct current and radio frequencyvoltages, only ions of a given mass-to-charge ratio will resonate andhave a stable trajectory to pass through the quadrupole and be detected.As used herein, “positive ion mode” refers to a mode wherein positivelycharged ions are detected by the mass analyzer, and “negative ion mode”refers to a mode wherein negatively charged ions are detected by themass analyzer.

As used herein selected reaction monitoring (SRM) refers to thetechnique of using tandem mass spectrometry to select and measure aparticular fragment ion of a selected precursor ion. For “selected ionmonitoring” or “SIM,” the amplitude of the direct current and the radiofrequency voltages are set to observe only a specific mass.

As used herein, the term multiple reaction monitoring (MRM) refers tothe technique of using tandem mass spectrometry to select and measuremore than one parent/precursor and fragment/product pairs within a givenanalysis. MRM is the application of SRM to multiple product ions fromone or more precursor ions.

The term “centrifugation” refers to a process that involves theapplication of the centripetal force for the sedimentation ofheterogeneous mixtures with a centrifuge. The increase the effectivegravitational force on a sample, for example, contained in a tube, tomore rapidly and completely cause the precipitate (pellet) to gather onthe bottom of the tube. The remaining solution is termed “supernatant.”

As used herein, the term “prostaglandin D2” or “PGD₂” refers to aprostaglandin that binds to the prostaglandin D₂ receptor (PTGDR) (DP₁)and the chemo attract an receptor-homologus molecule expressed on TH2cells (CRTH2) (DP₂).

As used herein, the term “accuracy” refers to closeness of the agreementbetween a test result and the accepted reference value expressed asabsolute and/or relative bias.

As used herein, the term “analyte” refers to a compound being measuredor detected and/or component represented in the name of a measurablequantity.

As used herein, the term “analytical measurement range” (AMR) refers tothe range of analyte values that a method can directly measure on thespecimen without any dilution, concentration, or other pretreatment notpart of the usual assay process.

As used herein, the term “analytic interferences” refers to anartifactual increase or decrease in apparent concentrations, activity,or intensity of an analyte due to the presence of a substance thatreacts specifically or nonspecifically with either the detection reagentor the signal itself.

As used herein, the term “interferences” refers to the influence of thepresence of hemolysis, lipemia, and icterus on the measurementprocedure's ability to accurately measure an analyte.

As used herein, the term “specificity” refers to the ability of themeasurement procedure to discriminate the analyte of interest whenpresented with substances potentially found within a sample. In anembodiment, it is expressed as a percent (%) cross-reactivity and/orresponse to substances other than analyte of interest in the absence ofthe analyte of interest.

As used herein, the term “selectivity” refers to the ability of themeasurement procedure to accurately measure the analyte of interestwithout contribution of the substances potentially found within asample. In an embodiment, it is expressed as a % cross-reactivity and/orresponse to substances other than analyte of interest in the presence ofthe analyte of interest.

As used herein, the term “maximum dilution” or “maximum concentration”or “clinical reportable range” refers to the established laboratoryspecifications for the maximum dilution and/or concentration that may beperformed to obtain a reportable numeric result.

As used herein, the term “Limit of Blank” (LOB) refers to the highestmeasurement result that is likely to be observed for a blank sample(with a stated probability). LOB is typically expressed as mean plus1.645×SD (or 2×SD) of blank measurements.

As used herein, the term “Limit of Detection” (LOD) refers to the lowestamount of analyte in a sample that can be detected (with statedprobability). LOD is typically expressed as LOB plus 1.645×SD (or 2×SD)of low sample measurements.

As used herein, the term “Lower Limit of Quantitation” (LLOQ) refers tothe lowest amount of analyte in a sample that can be quantitativelydetermined with stated acceptable precision and accuracy.

As used herein, the term “Upper Limit of Quantitation” (ULOQ) refers tothe highest amount of analyte in a sample that can be quantitativelydetermined without dilution.

As used herein, the term “Intra-run Imprecision” refers to the closenessof the agreement between the results of successive measurements of thesame measure and carried under the same conditions of measurements (sameanalytical run).

As used herein, the term “Inter-run Imprecision” refers to the closenessof the agreement between independent test results obtained understipulated conditions (different analytical runs and/or operators,laboratories, instruments, reagent lots, calibrators, etc.).

As used herein, the term “Reference Interval” refers to an intervalthat, when applied to the population serviced by the laboratory,correctly includes most of the subjects with characteristics similar tothe reference group and excludes the others.

As used herein, the term “biomarker” or “marker” refers to one or morenucleic acids, polypeptides and/or other biomolecules (e.g., PGD₂) thatcan be used to diagnose, or to aid in the diagnosis or prognosis of adisease or syndrome of interest, either alone or in combination withother biomarkers; monitor the progression of a disease or syndrome ofinterest; and/or monitor the effectiveness of a treatment for a syndromeor a disease of interest.

As used herein, the term “on-line” refers to purification or separationsteps that are performed in such a way that the test sample is disposed,e.g., injected, into a system in which the various components of thesystem are operationally connected and, in some embodiments, in fluidcommunication with one another.

In contrast to the term “on-line”, the term “off-line” refers to apurification, separation, or extraction procedure that is performedseparately from previous and/or subsequent purification or separationsteps and/or analysis steps. In such off-line procedures, the analytesof interests typically are separated, for example, on an extractioncolumn or by liquid/liquid extraction, from the other components in thesample matrix and then collected for subsequent introduction intoanother chromatographic or detector system. Off-line procedurestypically require manual intervention on the part of the operator.

Methods for the analysis of PGD₂ by LC-MS/MS

Embodiments of the present disclosure relate to methods and systems forthe measurement of prostaglandins. In an embodiment, the prostaglandinis prostaglandin (PGD₂). The present disclosure may be embodied in avariety of ways. The measurement of the prostaglandin, e.g., PGD₂ may beused for clinical diagnosis. In an embodiment, the disclosed methods andsystems allow for measurement of the prostaglandin, e.g., PGD₂, withoutthe need for derivatization processes. In certain embodiments, thebiological samples suitable for analysis by the methods and systems ofthe disclosure can include any sample that can contain the prostaglandinof interest. In an embodiment, PGD₂ or other prostaglandin is endogenousto a subject.

In one embodiment, the disclosure comprises a method for determining thepresence or amount of PGD₂ in a biological sample comprising: providinga biological sample believed to contain PGD₂; optionally,chromatographically separating PGD₂ from other components in the sample;and analyzing the chromatographically separated PGD₂ by tandem massspectrometry to determine the presence or amount of PGD₂ in thebiological sample. In some embodiments, the biological sample isobtained from a human or another mammal. In some instances thebiological sample is a urine sample. In other instances the biologicalsample is serum.

In certain embodiments, disclosed is a method for determining thepresence or amount of PGD₂ in a biological sample by tandem massspectrometry. The method may comprise any one of the steps of: (a)obtaining a biological sample from a subject; (b) optionally adding astable isotope-labeled PGD₂ to the biological sample as an internalstandard; (c) optionally performing solid phase extraction; (d)performing HPLC; and (e) measuring the PGD₂ (both labeled and unlabeled)by mass spectrometry. In certain embodiments, the mass spectrometry istandem mass spectrometry (MS/MS). For example, in one embodiment, thetandem MS/MS spectrometry comprises use of a triple quadrupole tandemmass spectrometer.

In an embodiment, the tandem mass spectrometry may comprise the stepsof: (i) generating a precursor ion of PGD₂; (ii) generating one or morefragment ions of the precursor ion; and (iii) detecting the presence oramount of the precursor ion generated in step (i) and/or the at leastone or more fragment ions generated in step (ii), or both, and relatingthe detected ions to the presence or amount of the PGD₂ in the sample.In an embodiment, the step of relating the detected ions to the presenceor amount of the PGD₂ in the sample is quantitative. In certainembodiments, the tandem mass spectrometry uses negative ion modeelectrospray ionization (ESI). Also, in certain embodiments,quantification of the analyte of interest (i.e., PGD₂) and the optionalinternal standard is performed in selected reaction monitoring mode(SRM).

In certain embodiments, the tandem mass spectrometry is coupled to HPLC.The HPLC step may directly precede the tandem mass spectrometry analysis(i.e., LC-MS/MS). In some embodiments, the HPLC is high turbulenceliquid chromatography (HTLC).

In some embodiments, a solid phase extraction is used to partiallypurify the PGD₂ prior to HPLC. Also in some embodiments, a second stableisotope labeled PGD₂ (i.e., a different isotope) is added to the sampleas an internal standard after the extraction but prior to the LC-MS/MS.In some embodiments, duplicate sets of charcoal stripped calibrators areanalyzed in each batch. The method may alternatively be used to measureother prostaglandins, e.g., PGI₂, PGE₂ or PGF and subtypes thereof.

In an embodiment, the LC-MS/MS is performed on-line. Thus, as disclosedherein, any one of the steps of the method may be controlled by acomputer. In some embodiments, the computer comprises one or more dataprocessors and/or a non-transitory computer readable storage mediumcontaining instructions (e.g. software program). Thus, also disclosedherein is a non-transitory computer readable storage medium containinginstructions which, when executed on one or more computers, cause theone or more computers to perform actions comprising at least one of thesteps of the methods disclosed herein.

The method may, in certain embodiments, comprise the measurement ofmultiple m/z precursor-fragment transitions. For example, in certainembodiments, and as explained in more detail herein, a first fragment isselected for quantitation of PGD₂, whereas an additional fragment orfragments may be chosen as a qualitative standard(s). In someembodiments, the method further comprises detection of an internalstandard (which as described herein may be added prior to the extractionsteps and/or prior to the HPLC/HTLC step). In some embodiments, theinternal standard is a stable isotope labeled PGD₂, such as PGD₂-d₉ orPGD₂-d₄ as discussed in more detail herein. Thus, in some embodiments,the internal standard is detected by: (i) generating a precursor ion ofPGD₂-d₉; (ii) generating one or more fragment ions of the precursor ion;and (iii) detecting the presence or amount of the precursor iongenerated in step (i) and/or the at least one or more fragment ionsgenerated in step (ii), or both, and relating the detected ions to thepresence or amount of the PGD₂-d₉ of the internal standard.

For example, for PGD₂, the transition of a precursor ion of about 351.3m/z to a fragment ion of about 233.1 m/z may be measured. For theinternal standard PGD₂-d₉, the transition of a precursor ion of about360.4 m/z to a fragment ion of about 232.9 m/z may be measured.

The internal standard may be used for qualitative and/or quantitativepurposes. For example, using isotope dilution mass spectrometry, astable isotope-labeled analogue of the analyte is added to the sample asan internal standard is measure concurrently with the analyte byLC-MS/MS. In some embodiments, the methods further comprise dual isotopedilution. In some embodiments the method comprise adding a first stableisotope labeled PGD₂ to the sample as an internal standard. In furtherembodiments, the method comprises adding a second stable isotope labeledPGD₂ to the sample as an internal standard. In certain instances, afirst isotope is added to the extraction step and a second isotope isassed after the extraction step, but prior to the LC-MS/MS. In someembodiments, the first isotope is PGD₂-d₄ ([2H4]PGD₂) and the second isPGD₂-d₉ ([2H9]PGD₂). In other embodiments, the first isotope is[2H9]PGD₂ and the second is [2H4]PGD₂. In some embodiments, theprecursor ion PGD₂-d₄ has a mass/charge ratio (m/z) of about 355.4 andthe one or more fragment ions for quantitation comprise a fragment ionwith a m/z of about 275.300, 237.300, 193.2, or 255.5.

Sample preparation can be used to simplify complex sample matrices,remove components, allow for analytes present at low concentration insamples to be concentrated, as well as facilitate solvent switching. Insome embodiments, the methods of the disclosure comprise at leastpartial purification of PGD₂ prior to LC-MS/MS. In some embodiments, themethods may comprise at least one purification step, such as proteinprecipitation, liquid-liquid extraction (LLE), solid phase extraction(SPE), immunopurification, and any combination thereof. In certainembodiments, the sample is subjected to an extraction column. In someembodiments, the column is a SPE column. In some instances, theextraction and/or mass spectrometry are performed on-line. The partialpurification method may also include sample dilution prior to analysisby LC-MS/MS.

The method may also comprise the use of calibration standards. In anembodiment, such standards are run through each of the steps of themethod so as to correct for sample loss during any step. In someembodiments, duplicate sets of charcoal stripped calibrators areanalyzed in each batch. The back-calculated amount of the individualanalyte in the sample may then be determined from calibration curvesgenerated by spiking known amounts of each purified analyte intocharcoal stripped urine or serum to generate a final concentration ofpurified analyte of interest. In an embodiment, the calibration standardare purified PGD₂ that is within the range of about 1.0 to 1,000 pg/mL.Or, narrower or larger ranges may be used.

An example of a method (2) of the present disclosure is shown in FIG. 1.Thus, in an embodiment, the method may include a step of providing asample, for example, a serum or urine sample believed to contain PGD₂(4). In some embodiments, an appropriate internal standard is added tothe sample (6). For example, in some embodiments for analyzing PGD₂ inbiological samples, at least one of PGD₂-D₉ is added as an internalstandard for the measurement of PGD₂. Or, other stable labeled isotopesof PGD₂ may be used.

In some embodiments, the analyte of interest (i.e., PGD₂) is partiallypurified by solid phase extraction (SPE) (8) of the biological sampleprior to HPLC. Additionally and/or alternatively, the sample may bediluted in a solvent that can be used for LC or MS in subsequentpurification steps. In an embodiment, the SPE is used to concentrate andpartially purify the analyte. For example, the SPE may removephospholipids and/or fibrinogen from the biological samples. In someembodiments, the SPE is a polymeric SPE sorbent combined with matrixremoval. After SPE, the sample can be centrifuged (e.g., 2000 rpm or1207 g) for about 1 minute, the supernatant decanted, and the pelletedsample evaporated to remove residual solvent and then reconstituted in asolvent appropriate for LC or HPLC (e.g., acetonitrile:water).

Still referring to FIG. 1, the method may further include liquidchromatography (9) as a means to separate the analyte of interest fromother components in the sample. In an embodiment, two liquidchromatography steps are used. For example, the method may comprise afirst extraction column liquid chromatography followed by transfer ofthe biomarker of interest to a second HPLC analytical column. In otherembodiments, only one HPLC step is used. In some embodiments, HTLC isused.

For example, the reconstituted extract may be applied onto a HPLC orHTLC system, wherein the analytes are eluted using an isocraticseparation through an extraction column. In certain embodiments, themobile phase that is used comprises a gradient.

The LC may, in certain embodiments, comprise high turbulence liquidchromatography or high throughput liquid chromatography (HTLC)(sometimes referred to as turbulent flow liquid chromatography (TFLC).In some embodiments, HTLC, alone or in combination with one or morepurification methods, may be used to purify the biomarker of interestprior to mass spectrometry. Also, in some embodiments, the use of a HTLCsample preparation method can eliminate the need for other samplepreparation methods including SPE. Thus, in some embodiments, the testsample, e.g., a biological fluid, can be disposed, e.g., injected,directly onto a high turbulence liquid chromatography system.

For example, in one embodiment, an Aria TX4 HTLC System (ThermoScientific MA) consisting of 4-1100 Series Quaternary Pumps, 4-1100Series Binary Pumps, 8-1100 Series Vacuum Degasser or 8-1200 SeriesBinary Pumps, 8-1200 Series Vacuum Degasser is used. In this embodiment,the sample is reconstituted in 10% acetonitrile in reagent grade waterprior to application to the HTLC column. In an embodiment, the Pump Amobile phase is 0.1% formic acid in water and the Pump B mobile phase is100% acetonitrile.

The separated analytes are then introduced into a mass spectrometer (MS)system (10). In some embodiments, a tandem MS/MS system is used. In anembodiment, an API 4000, API 5000, or API 5500 (or equivalent) TandemMass Spectrometer, Danaher (Toronto, CA) is used. The analyte ofinterest (i.e., PGD₂) may then be quantified based upon the amount ofthe characteristic transitions measured by tandem MS as detailed herein.In some embodiments, the tandem mass spectrometer comprises a triplequadrupole mass spectrometer.

In mass spectrometry, analytes are ionized to produce gas phase ionssuitable for resolution in the mass analyzer. Ionization occurs in theion source. There are several ion sources known in the art. In someembodiments, the analyte may be ionized by any method known in the art.For example, ionization may be performed using any of the following ionsources: atmospheric pressure chemical ionization (APCI), atmosphericpressure photoionization (APPI), electron impact ionization (EI),electrospray ionization (ESI), matrix assisted laser desorption (MALDI),surface enhanced laser desorption ionization (SELDI), thermosprayionization, inductively coupled plasma (ICP), and fast atom bombardment(FAB). PGD₂ may be ionized in positive or negative ion mode. In oneembodiment, ESI is used.

After the sample has been ionized, the charged ions may be analyzed todetermine mass-to-charge ratios (m/z). For example, quadrupole massspectrometers, ion trap mass spectrometers, and time-of-flight (TOF)mass spectrometers can be used to produce a mass spectra for an analyteof interest. Ions may be detected using any detection mode generallyknown in the art, including but not limited to selective ion monitoring(SIM), multiple reaction monitoring (MRM), and selected reactionmonitoring (SRM).

In certain embodiments, the mass spectrometer uses a “quadrupole”system. In a “quadrupole” or “quadrupole ion trap” mass spectrometer,ions in an oscillating radio frequency (RF) field experience a forceproportional to the direct current (DC) potential applied betweenelectrodes, the amplitude of the RF signal, and m/z. The voltage andamplitude can be selected so that only ions having a particular m/ztravel the length of the quadrupole, while all other ions are deflected.Thus, quadrupole instruments can act as both a “mass filter” and as a“mass detector” for the ions injected into the instrument.

In an embodiment, the methods and systems of the present disclosure usea triple quadrupole MS/MS (see e.g., Yost, Enke in Ch. 8 of Tandem MassSpectrometry, Ed. McLafferty, pub. John Wiley and Sons, 1983). Triplequadrupole MS/MS instruments typically consist of two quadrupole massfilters separated by a fragmentation means. Quadrupole 1 (Q1) is a massfilter that allows for selection of precursor ions and Q3 allows forselection of product ions based on mass-to-charge ratios. Quadrupole 2(Q2) is the collision cell where the precursor ions selected in Q1 arefragmented into product ions. While in Q2, precursor ions are collidedwith neutral molecules such as argon, nitrogen, or helium causing theprecursor ions to fragment in process called collision-induceddissociation (CID). The fragments are then accelerated into the thirdquadrupole (Q3) mass filter, which scans through the mass range andproduces a mass spectrum as the fragment ions hit a detector.

In certain embodiments, tandem mass spectrometry is used. See, e.g.,U.S. Pat. No. 6,107,623, entitled “Methods and Apparatus for Tandem MassSpectrometry,” which is hereby incorporated by reference in itsentirety. The selectivity of the MS technique can be enhanced by using“tandem mass spectrometry,” or “MS/MS.” MS/MS methods are useful for theanalysis of complex mixtures, especially biological samples, in partbecause the selectivity of MS/MS can minimize the need for extensivesample clean-up prior to analysis.

In one embodiment, the instrument may comprise a quadrupole mass filteroperated in the RF only mode as an ion containment or transmissiondevice. In an embodiment, the quadrupole may further comprise acollision gas at a pressure of between 1 and 10 millitorr. Many othertypes of “hybrid” tandem mass spectrometers are also known, and can beused in the methods and systems of the present disclosure includingvarious combinations of magnetic sector analyzers and quadrupolefilters. These hybrid instruments often comprise high resolutionmagnetic sector analyzers (i.e., analyzers comprising both magnetic andelectrostatic sectors arranged in a double-focusing combination) aseither or both of the mass filters. Use of high resolution mass filtersmay be highly effective in reducing chemical noise to very low levels.

The mass spectrometer provides the user with an ion scan displaying therelative ion abundance (peaks) of the ions in the mass spectrum (12).Thus, the mass spectrum can be used to determine the amount of analyte(i.e., PGD₂) present in the sample (14). In some instances, theback-calculated amount of each analyte in each sample may be determinedby comparison of the sample response or response ratio when employinginternal standardization to calibration curves generated by spiking aknown amount of purified analyte material into a standard test sample,e.g., charcoal stripped human urine. In one embodiment, calibrators areprepared at known concentrations to generate a response or responseratio when employing internal standardization versus concentrationcalibration curve. In an embodiment, this determination is performed atleast in part by a computer or data analysis system and/or anon-transitory computer readable storage medium containing instructionswhich, when executed on the one or more data processors, cause the oneor more data processors to perform actions to make this determination.

In various embodiments, the method includes a detailed review of rawdata, quality control, review and interpretation of patient resultsfollowed by release to the laboratory system. For example, in certainembodiments, duplicate calibration curves are used for each batch ofsamples. In certain embodiments, a total of 25% of standard points maybe excluded from the combined curves if the back-calculatedconcentrations exceed the theoretical concentrations by >20% at the LLOQor >15% at other concentrations. Or, other cut-offs may be used. In anembodiment, a result may be reported below the lowest, or above thehighest remaining standard. In an embodiment, the standard curvecorrelation coefficient is (r)>0.98. Also, in certain embodiments, themethod requires that control pools be within acceptable limits as isknown in the art. In an embodiment, all chromatographic peak shapes arereviewed for consistency. For example, where peak distortion isobserved; a contaminant may be present. In an embodiment, the methodincludes ensuring that the correct peak is integrated where multiplepeaks are observed within the chromatogram by confirming that theretention time of the peak integrated corresponds to calibrators andquality control samples. For example, the method may include review ofthe internal standard peak area vs. mass spectrum index plot. In anembodiment, internal standard peak areas more than 50% greater than theneighboring peaks may be submitted for repeat analysis and/or internalstandard peak areas more than 33% less than the neighboring peaks may besubmitted for repeat analysis. Or, other cut-offs may be used. In anembodiment, this review and analysis is done by a computer or dataanalysis system and/or a non-transitory computer readable storage mediumcontaining instructions, which when executed on the one or more dataprocessors, cause the one or more data processors to perform actions toperform this analysis and/or review.

Systems for the Analysis of PGD₂

Also disclosed are systems for determining the presence or amount of aprostaglandin, e.g., PGD₂, in a sample. For example, in some embodimentsthe system may comprise: a station or component for providing abiological sample believed to contain a prostaglandin of interest suchas PGD₂; optionally a station or stations (or component(s)) for sampleextractions (i.e., clean-up or prepurification) and/orchromatographically separating the prostaglandin (e.g., PGD₂) from othercomponents in the biological sample; and a station or component foranalyzing the chromatographically separated prostaglandin (e.g. PGD₂) bymass spectrometry to determine the presence or amount of theprostaglandin (e.g., PGD₂) in the biological sample. In an embodiment,the sample is a biological sample obtained from a human or anothermammal. For example, the sample may be human serum or urine.

In an embodiment, the mass spectrometry is tandem mass spectrometry(MS/MS). In an embodiment, the mass spectrometry is operated inelectrospray ionization (ESI) mode. In an embodiment, quantification ofPGD₂ is performed in selected reaction monitoring mode (SRM). Forexample, the station for tandem mass spectrometry may comprise a SciexAPI4000, API5000, or API5500 tandem mass spectrometer (or equivalent),Danaher (Toronto, CA).

In one embodiment, the station for chromatographic separation comprisesat least one component, e.g., apparatus, to perform liquidchromatography (LC). In one embodiment, the station for liquidchromatography comprises a column for extraction chromatography.Additionally or alternatively, the station for liquid chromatographycomprises a column for analytical chromatography. In certainembodiments, the column for extraction chromatography and analyticalchromatography comprise a single station or single column. Variouscolumns comprising stationary phases and mobile phases that may be usedfor extraction or analytical LC are described herein. In someembodiments, the extraction column is a functionalized silica orpolymer-silica hybrid or polymeric particle or monolithic silicastationary phase. In some embodiments, a core-shell silica solid supportis use. A column used for analytical liquid chromatography may be varieddepending on the column that was used for the extraction liquidchromatography step. For example, in certain embodiments, the analyticalcolumn comprises particles having an average diameter of about 5 μm. Inother embodiments, the analytical column comprises particles having anaverage diameter of about 2.6 μm. In some embodiments, the analyticalcolumn is a functionalized silica or polymer-silica hybrid, or apolymeric particle or monolithic silica stationary phase. Thus, in someembodiments, the stationary phase is C18 with TMS endcapping. Forexample, in some embodiments the LC column is a Phenomenex Kinetex 2.6μm C18(2) 100 Å, 150×4.6 mm.

In some embodiments, HPLC is used to purify the PGD₂ from othercomponents in the sample that co-purify with the PGD₂ after extractionand/or dilution of the sample. Or, in other embodiments, HTLC is used topurify the PGD₂ from other components in the sample. For example, in oneembodiment, an Aria TX4 HTLC System (Thermo Scientific MA) consisting of4-1100 Series Quaternary Pumps, 4-1100 Series Binary Pumps, 8-1100Series Vacuum Degasser or 8-1200 Series Binary Pumps, 8-1200 SeriesVacuum Degasser is used.

In an embodiment, the system may further comprise a station or componentfor partially purifying the PGD₂ from other components in the sample asfor example by liquid-liquid extraction (LLE) and/or dilution. Or, insome embodiments, solid phase extraction (SPE) may be used. Thus, incertain embodiments, the system may also comprise a station forextracting the PGD₂ from the biological sample and/or diluting thesample. The station for partial purification (e.g., SPE) may compriseequipment and reagents for addition of solvents to the sample andremoval of waste fractions. In some cases a isotopically-labeledinternal standard such as PGD₂-d₉ (SantaCruz—SC-224218) is used tostandardize losses of the biomarker that may occur during theprocedures. Thus, the station for SPE may comprise a hood or othersafety features required for working with solvents and/or isotopes.

Also, in certain embodiments, at least one of the stations is automatedand/or controlled by a computer. For example, as described herein, incertain embodiments, at least some of the steps are automated such thatlittle to no manual intervention is required. For example, as disclosedherein, any one of the stations or components may be controlled by adata processor or a computer. Also disclosed herein is a data processorand/or a non-transitory computer readable storage medium containinginstructions (i.e., software) which, when executed on the one or moredata processors or computers, cause the one or more data processors orcomputers to perform actions for at least one of the stations of thesystem.

FIG. 2 shows an embodiment of a system (200) of the present invention.As shown in FIG. 2, the system may comprise a station for aliquoting asample (202) that may comprise a biomarker (e.g., PGD₂) of interest intosampling containers. In one embodiment, the sample is aliquoted into acontainer or containers to facilitate SPE or sample dilution. Thestation for aliquoting may comprise receptacles to discard the portionof the sample that is not used in the analysis. The station foraliquoting a sample (202) may further comprise a station for adding aninternal standard to the sample. In an embodiment, the internal standardcomprises the biomarker (e.g., PGD₂) of interest labeled with anon-natural isotope. Thus, the station for adding an internal standardmay comprise safety features to facilitate adding an isotopicallylabeled internal standard solutions to the sample. The system may also,in some embodiments, comprise a station (206) for sample clean-up (i.e.,purification of the analyte away from other components in the sampleand/or dilution of the sample). In some embodiments, the station forsample clean-up comprises a station for SPE, liquid-liquid extraction,protein precipitation, dilution of the samples or other types ofpurification procedures.

The system may also comprise a station for liquid chromatography (LC) ofthe sample (108). As described herein, in an embodiment, the station forliquid chromatography may comprise an extraction liquid chromatographycolumn, or the station may comprise HPLC and no extraction column. Or,as discussed in more detail below, other types of liquid chromatography,such as high turbulence liquid chromatography (HTLC) may be used. Forexample, in one embodiment, an Aria TX4 HTLC System (Thermo ScientificMA) consisting of 4-1100 Series Quaternary Pumps, 4-1100 Series BinaryPumps, 8-1100 Series Vacuum Degasser or 8-1200 Series Binary Pumps,8-1200 Series Vacuum Degasser is used. In this embodiment, the samplemay be reconstituted in 10% acetonitrile in reagent grade water prior toapplication to the HTLC column. In an embodiment, the Pump A mobilephase is acetonitrile:methanol:water (5:5:90) and the Pump B mobilephase is acetonitrile:methanol:water (45:45:10).

Thus, the station for liquid chromatography may comprise a columncomprising the stationary phase, as well as containers or receptaclescomprising solvents that are used as the mobile phase. The station maycomprise the appropriate lines and valves to adjust the amounts ofindividual solvents being applied to the column or columns. Also, thestation may comprise a means to remove and discard those fractions fromthe LC that do not comprise the biomarker of interest. In an embodiment,the fractions that do not contain the biomarker of interest arecontinuously removed from the column and sent to a waste receptacle fordecontamination and to be discarded.

Also, the system may comprise a station for characterization andquantification of the PGD₂ biomarker (FIG. 2). In one embodiment, thesystem may comprise a station for mass spectrometry (MS) of the PGD₂biomarker(s) (210). In an embodiment, the station for mass spectrometrycomprises a station for tandem mass spectrometry (MS/MS). The system mayfurther comprise a station (212) for instrument control and dataanalysis, wherein the station interacts with the station (206) forsample clean-up, the station (208) for LC, and/or the station (210) forMS. Also, the station for instrument control and data analysis mayfurther comprise a stations for characterization and quantification(212). The station (212) for data analysis may be part of the MS/MSstation or a separate station and may comprise a computer and/orsoftware for analysis of the MS/MS results. In an embodiment, thestation (212) for instrument control and/or data analysis comprises acomputer or data processor and/or a non-transitory computer readablestorage medium (e.g., software) containing instructions, which whenexecuted on the one or more data processors, cause the one or more dataprocessors to perform the data analysis. In an embodiment, the analysiscomprises both identification and quantification of the biomarker ofinterest (e.g., PGD₂). In an embodiment, the analysis system comprises acomputer (300).

FIG. 3 shows a block diagram of a computer 300 used for detection and/orquantification of a biomarker of interest (e.g., PGD₂). As illustratedin FIG. 3, modules, engines, or components (e.g., program, code, orinstructions) executable by one or more processors may be used toimplement the various subsystems of an analyzer system according tovarious embodiments. The modules, engines, or components may be storedon a non-transitory computer medium. As needed, one or more of themodules, engines, or components may be loaded into system memory (e.g.,RAM) and executed by one or more processors of the analyzer system. Inthe example depicted in FIG. 3, modules, engines, or components areshown for implementing the methods or running any of the systems of thedisclosure.

Thus, FIG. 3 illustrates an example computing device 300 suitable foruse with systems and the methods according to this disclosure. Theexample computing device 300 includes a processor 305 which is incommunication with the memory 310 and other components of the computingdevice 300 using one or more communications buses 315. The processor 305is configured to execute processor-executable instructions stored in thememory 310 to perform one or more methods or operate one or morestations for detecting biomarker of interest (e.g., PGD₂) according todifferent examples. In this example, the memory 310 may storeprocessor-executable instructions 325 that can analyze 320 results forsample as discussed herein.

The computing device 300 in this example may also include one or moreuser input devices 330, such as a keyboard, mouse, touchscreen,microphone, etc., to accept user input. The computing device 300 mayalso include a display 335 to provide visual output to a user such as auser interface. The computing device 300 may also include acommunications interface 340. In some examples, the communicationsinterface 340 may enable communications using one or more networks,including a local area network (“LAN”); wide area network (“WAN”), suchas the Internet; metropolitan area network (“MAN”); point-to-point orpeer-to-peer connection; etc. Communication with other devices may beaccomplished using any suitable networking protocol. For example, onesuitable networking protocol may include the Internet Protocol (“IP”),Transmission Control Protocol (“TCP”), User Datagram Protocol (“UDP”),or combinations thereof, such as TCP/IP or UDP/IP.

In some embodiments, one or more of the purification or separation stepscan be performed “on-line.” The on-line system may comprise anautosampler for removing aliquots of the sample from one container andtransferring such aliquots into another container. For example, anautosampler may be used to transfer the sample after extraction onto anLC extraction column. The on-line system may comprise one or moreinjection ports for injecting the fractions isolated from the LCextraction columns onto the LC analytical column and/or one or moreinjection ports for injecting the LC purified sample into the MS system.Thus, the on-line system may comprise one or more columns, including butnot limited to an HTLC column. In such “on-line” systems, the testsample and/or analytes of interest can be passed from one component ofthe system to another without exiting the system, e.g., without havingto be collected and then disposed into another component of the system.

In some embodiments, the on-line purification or separation method ishighly automated. In such embodiments, the steps can be performedwithout the need for operator intervention once the process is set-upand initiated. For example, in one embodiment, the system, or portionsof the system (e.g., HTLC, MS/MS and data analysis) may be controlled bya computer. Thus, in certain embodiments, the system may comprisesoftware for controlling the various components of the system, includingpumps, valves, autosamplers, and the like. Such software can be used tooptimize the extraction process through the precise timing of sample andsolute additions and flow rate.

For example, FIG. 4 shows an embodiment, of an on-line plumbing diagramin which eluting pumps (402) are coupled to an LC column (404), which iscoupled to a selector valve (406), which is coupled to the massspectrometer (408).

Although some or all of the steps in the method and the stations orcomponents comprising the system may be on-line, in certain embodiments,some or all of the steps may be performed “off-line.”

Thus, the disclosure provides methods and systems for applying liquidchromatography and mass spectrometry as a means to separate a biomarkeranalyte of interest, such as PGD₂, from other components that may bepresent in a sample. The methods and systems may comprise an off-lineliquid-liquid extraction and/or sample dilution step as a means topartially purify the sample prior to HTLC and tandem mass spectrometry.The methods and systems may be used for clinical diagnosis.

The systems and methods may, in certain embodiments, provide for amultiplexed assay. For example, certain embodiments of the presentinvention may comprise a multiplexed liquid chromatography tandem massspectrometry (LC-MS/MS) or two-dimensional or tandem liquidchromatography-tandem mass spectrometry (LC)-LC-MS/MS) methods for thequantitative analysis of PGD₂ is biological samples.

Embodiments may provide certain advantages. In an embodiment, anaccurate, precise, simple and fast HPLC-MS/MS isotope dilutioncommercially available method has been developed to allow quantitativemeasurements of PGD₂ in urine or serum. Reference intervals can bedeveloped for adult men, adult women, and pediatric subjects. Also, inan embodiment, good correlation with other assay systems will allowresult interpretation for clinical conditions, including mastocytosisusing published data.

In certain embodiments, the methods and systems may provide greatersensitivity than the sensitivities previously attainable for PGD₂. Also,embodiments of the methods and systems may provide for rapid throughputthat has previously not been attainable for many of the analytes beingmeasured.

As another advantage, the specificity and sensitivity provided by thedisclosed methods and systems may allow for the analysis of analytesfrom a variety of materials. For example, the disclosed methods andsystems can be applied to the quantification of analytes of interest incomplex sample matrices, including, but not limited to urine or serum.Also, using the disclosed methods and systems allows for measurement ofPGD₂ without derivatization and at levels as low as 1 pg/mL. Thus, themethods and systems are suitable for clinical applications and/orclinical trials.

As additional potential advantages, in certain embodiments, thedisclosed systems and methods provide approaches for addressing isobaricinterferences, varied sample content, including hemolysed and lipemicsamples, while attaining low pg/mL limits of quantification (LLOQ) ofthe target analytes. Accordingly, embodiments of the disclosed methodsand systems may provide for the quantitative, sensitive, and specificdetection of clinical biomarkers used in clinical diagnosis. Forexample, in some embodiments the lower limit of detection using a samplealiquot of 400 μL is at least 10 pg/mL, 5 pg/mL, 2 pg/mL, or 1 pg/mL. Incertain embodiments, the lower limit of detection using a sample aliquotof 500 μL is at least 10 pg/mL, 5 pg/mL, 2 pg/mL, or 1 pg/mL.

Embodiments of the methods and systems of the present disclosure mayprovide for rapid throughput that has previously not been attainable formany of the analytes being measured. For example, using the methods andsystems of the present disclosure, multiple samples may be analyzed forPGD₂ using 96 well plates and a multiplex system of four LC-MS/MSsystems, significantly increasing the throughput.

EXAMPLES

The following examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter.

Example 1. Measurement of PGD₂ in Urine by SPE-LC-MS/MS

Specimens. All studies were performed using urine samples. Acceptablesample volumes are shown in TABLE 1. The minimum sample volume wasdetermined to be 1 mL of urine.

TABLE 1 Specimen Collection Adult: 5 mL urine is acceptable Pediatric:2.5 mL urine is acceptable Minimum: 1 mL urine is acceptable

Urine samples were collected into plastic containers. Specimens werestored according to the following conditions:

-   -   Short Term Storage        -   Urine Frozen (≤−10° C. and ≤−55° C.): 8 days        -   Urine Refrigerated (2-8° C.): 3 days        -   Urine Room Temperature (15-30° C.): 3 days        -   Urine Freeze/Thaw (≤−10° C.): 6 Cycles    -   Long Term Storage        -   Urine Frozen (≤−10° C.): Stability testing in process    -   Shipping Conditions:        -   Urine Frozen (≤−10° C.): - on dry ice

PGD₂ was stable for at least 3 days at 4° C. and at room temperature and8 days frozen (≤−10° C. or ≤−55° C.). PGD₂ was stable for at least 6freeze-thaw cycles (≤−10° C. or ≤−55° C.).

Assay parameters. The assay was conducted according to the parametersprovided in TABLE 2.

TABLE 2 Assay parameters Parameter Value or Criteria Reportable RangeAMR Maximum Dilution/ Units of (LLOQ-ULOQ) Concentration MeasureProstaglandin D₂ 1-1000 pg/mL X10/10000 pg/mL pg/mL Max Dilution Reducedvolume of 40 μL is allowable. Preferred Specimen Urine

Acceptance criteria. For acceptance, no more than 25% of all standardcurve values may be deleted. Previous standard curve data may be used toidentify standard points to delete. Standard curve regression was basedupon individual replicates, and each replicate was considered a pointfor acceptance rules.

Controls. Three levels of control pools were prepared from human urinepools, spiked or diluted is required to meet target values. For very lowquality control (QC) levels that were not feasible to use human urinematrix, charcoal stripped human urine was used as a diluent for thehuman urine pools.

A control result was defined as the mean of two duplicates withcoefficient variation (CV)<20%. The mean of both duplicates was used indetermining acceptance.

Dilutions. Sample dilutions were made with standard matrix, with amaximum allowable dilution factor of ×10. The recommended dilutionscheme is shown in TABLE 3.

TABLE 3 Recommended Dilution Scheme Dilution Volume of Volume of LevelSample Sample (μL) Diluent, S0 (μL) X10 Neat Human Urine 20 180

Sample preparation. Frozen standards, controls, and samples were thawedand vortexed three times. An aliquot (e.g., 400 μL) of the standard,control, or sample (neat or diluted) were pipetted into 12×75 mmborosilicate glass tubes. When sufficient volume was unavailable,samples were added in lower amounts. The acceptable volume for reducedvolume analysis was determined to be at least 40 μL. 100 μL of internalstandard was pipetted into each tube, except for four tubes that wereleft empty as double blanks. The glass tubes were covered with parafilmand vortexed 10 times. 1.2 mL of 1% Formic Acid in water was added toall tubes, including the double blanks. The glass tubes were coveredwith parafilm and vortexed 10 times. The appropriate dilution factor wasapplied to reduce volume analyses.

SPE plate processing. A Strata X Pro 96-well plate (Phenomenex Catalog#8E-5536-TGA, or equivalent) was placed on top of a waste collectionplate. 1 mL of methanol was added to each well and the wells weredrained by gravity without the use of positive pressure. 1 mL of 5%methanol in water was then added to each of the wells drained by gravitywithout the use of positive pressure. The entire volume of dilutedstandards, controls, and samples were transferred to the correspondingwells of the plate. The wells were first drained by gravity and thenpositive pressure was applied on low setting. 1 mL of 5% methanol inwater was added to each of the wells of the 96-well plate and the wellswere drained by gravity and then positive pressure was applied on lowsetting. 1 mL of 20% methanol in water with 2% ammonium hydroxide wasthen added to each of the wells and the wells drained by gravity andthen positive pressure was applied on low setting. Next, 1 mL of 5%methanol in water was added to each of the wells and after the wellswere drained by gravity, positive pressure was applied on low setting.Next, 1 mL of 30% methanol in water with 2% formic acid was added toeach of the wells and after the wells were drained by gravity, positivepressure was applied on low setting. 1 mL of 5% methanol in water wasthen added to each of the wells and the wells drained by gravityfollowed by positive pressure applied on low setting. Next, 1 mL of 40%methanol in water was added to each of the wells and the wells weredrained by gravity and then positive pressure was applied on lowsetting. The positive pressure setting was switched to high for 1minute. Next, the plate was placed on top of a new 1.2 mL 96-wellcollection plate and 300 μL of 2% acetic acid in chloroform was added toeach of the wells of the sample/standard plate. The wells were drainedby gravity and then positive pressure was applied on low setting. Again,300 μL of 2% acetic acid in chloroform was added to each of the wells.The wells were again drained by gravity, and then positive pressure wasapplied on low setting. The 1.2 mL 96-well plate with collected sampleswas then placed into a TurboVap 96 Concentration Workstation (BiotageLife Sciences) for approximately 30 mins at 40° C. until samples weredry. Then, 120 μL of PGD₂ reconstitution solution (3.33 ng/mL PGD₂-d₄internal standard in 1:3 methanol:10 mM ammonium acetate) was added toall wells, the plate was sealed, and mixed four times for 30 seconds(for a total of 2 minutes). The 96-well plate was then positioned in theLC-MS/MS Autosampler.

HTLC-MS/MS Procedure. For HTLC purification of PGD₂, a PhenomenexKinetex 2.6 μm C18(2) 100 Å, 150×4.6 mm column was used. All LC systemreagents were filled and LC pumps were primed to remove any bubbles frommobile phase lines or to remove mobile phase from previous assays. Themass spectrometer was then equilibrated for 1 minute. The Aria system(Aria OS Version 1.4 or greater, Cohesive Technologies (MA, USA)) wasstarted. Aria TX4 HTLC System, Cohesive Technologies, (MA, USA)consisting of 4 each: 1100 Series Quaternary Pump, 1100 Series BinaryPump, 1100 Series Vacuum Degasser, or 8 Series 1200 Binary Pump and 4Series 1200 Vacuum Degasser were started and primed at 5 mL per minutefor 5 minutes for each solvent to be used. Test injections wereperformed using UPGD₂ system suitability test (SST). The loading pumpwas run using a gradient starting with 60% mobile phase A (0.1% formicacid in water) and 40% mobile phase B (100% acetonitrile) at a flow rateof 0.8 mL/min. The eluting pump was run with 100% mobile phase A and 0%mobile phase B at a flow rate of 0.00 mL/min.

For MS/MS, an AB SCIEX API5000 triple quadrupole mass spectrometer,operating in negative ion electrospray ionization (ESI) mode(Turboionspray) was used for detection. Quantification of analyte andinternal standard was performed in selected reaction monitoring mode(SRM) with the use of ion summing. For PGD₂ the 351.3→233.1 transitionwas monitored. For the internal standard (PGD₂-d₉), the 360.4→232.9transition was monitored. A resulting MS/MS scan of the PGD₂ systemsuitability test (SST) is shown in FIG. 5. For FIG. 5, PGD₂-H₂O peaks of351.300 and 333.100 and PGD₂ SST peaks of 351.298 and 233.100 wereanalyzed. The signal:noise (S/N) ratio was 5.5. The peak intensity was7.7e+2 counts per second (cps) with a Ymax of 2.2e+2 cps and Ymin of8.0e+1 cps.

For detection of multiple product ions from one or more precursor ionsthe mass spectrometer was operated in multiple reaction monitoring (MRM)mode. The following transitions were monitored for each of the analyteslisted below:

-   -   PGD2-H2O: 351.300→333.100    -   d9-PGD2-H2O: 360.400→342.200    -   d4-PGD2-H2O: 355.400→337.000    -   PGD2-2H2O: 351.300→315.300    -   d9-PGD2-2H2O: 360.400→324.400    -   d4-PGD2-2H2O: 355.400→319.200    -   PGD2: 351.3→271.2; 351.298→271.200; 351.299→271.200;        351.301→271.200; 351.302→271.200; 351.299→233.100;        351.298→233.1; 351.301→233.100; 351.302→233.100;        351.300→189.100; 351.298→189.100; 351.299→189.100;        351.301→189.100; 351.302→189.100; 351.300→251.200;        351.298→251.200; 351.299→251.200; 351.301→251.200;        351.302→251.200    -   d9-PGD2: 360.400→280.100; 360.400→232.900; 360.400→189.000;        360.400→250.900    -   d4-PGD2: 355.400→275.300; 355.400→237.300; 355.400→193.201;        355.400→255.500

Calculations. Integration parameters were set using the QuantitationWizard in the Analyst Version 1.4 or greater. Sciex, (CA, USA)) program.A standard curve was generated and a metric plot was generated withindex on the x-axis vs. the internal standard on the y-axis. Standardcurves were used to determine the amount of PGD₂ present in each sample.Duplicate calibration curves were used for each batch of samples. Atotal of 25% of standard points may be excluded from the combined curvesif the back-calculated concentrations exceed the theoreticalconcentrations by >20% at the LLOQ or >15% at other concentrations. Noresult was reported below the lowest, or above the highest remainingstandard. Samples with values less than the minimum reportable dose werecalculated and reported as “less than” value. All chromatographic peakshapes were reviewed for consistency. Observation of peak distortionindicated the presence of a contaminant. Where multiple peaks wereobserved within the chromatogram, to ensure the correct peak wasintegrated, the retention of the peak integrated was confirmed tocorrespond to calibrators and quality control samples. Internal standardpeak areas vs. index plot were visually reviewed for gross indicationsof processing and/or technical errors. All internal standard peak areasthat visually appeared to be >50% of the neighboring peaks wereconsidered for repeat analysis. All internal standard peak areas thatvisually appeared to be 33% less than the neighboring peaks wereconsidered for repeat analysis. Such anomalies are good indicators ofreagent addition or pipetting errors or technical malfunctions ofequipment.

Example 2. Validation of the Measurement of PGD2 in Urine bySPE-LC-MS/MS

Standard Material. The standard lots were prepared by diluting thematerial from two separate vials of commercially available ProstaglandinD2 MaxSpec Standard purchased from Cayman Chemical Company into a poolof Mass Spect Gold Urine (GoldenWest BioSolutions) with 3.6 μg/mLindomethacin added for stability. The calibration standards used forvalidation ranged in concentration from 1-1000 pg/mL.

Controls. Clinical Quality Control pools used during this validationwere prepared by diluting commercially available Prostaglandin D2MaxSpec Standard purchased from Cayman Chemical Company into human urinepools. Three control pools were used in each validation batch.

Test procedures. The assay steps were performed according to UrinaryProstaglandin D2 by SPE and LCMS, as described in Example 1. Thevalidation was completed in 10 independent assays (one of which wasanalyzed twice for establishment of autosampler stability).

Acceptance Criteria. The acceptance criteria for each of the followingvalidation assays are shown in TABLE 4.

TABLE 4 Acceptance Criteria PARAMETER MATERIAL ACCEPTANCE CRITERIAIntra-assay Five levels of Prostaglandin D2 diluted in Runs include anLLOQ Standard charcoal stripped human urine at LLOQ, calibrator levelAccuracy and low, mid, high, and ULOQ target Exempt from QC acceptancePrecision concentrations. Preparation of accuracy and criteria precisionsamples was independent of Bias ≤ ± 15%; (LLOQ ± 20%) standardpreparation. Twenty replicates of CV ≤ 15%; (LLOQ ≤ 20%) each level wereanalyzed in a single batch. Inter-assay The above samples were analyzedin six Runs include an LLOQ Standard batches, with a minimum of threereplicates calibrator level Accuracy and at each level. Batch processingwas Exempt from QC acceptance Precision performed using differentreagent lots as criteria available. Mean inter-assay bias ≤ ± 15%; (LLOQ± 20%) and at least 2/3 of intra-assay bias values ≤ ± 15% CV ≤ 15%;(LLOQ ≤ 20%) and at least 2/3 of intra-assay CV values within rangeIntra-assay Human urine with high, medium and low CV ≤ 15%; (LLOQ ≤ 20%)Sample concentrations of Prostaglandin D2. Six Precision replicates ofeach level was analyzed in a single batch. Inter-assay The above sampleswere analyzed three CV ≤ 15%; (LLOQ ≤ 20%) and Sample times on differentdays with different lots at least 2/3 of intra-assay CV Precision ofreagent if possible. values within range Lower Limit of Prostaglandin D2diluted in charcoal Lowest concentration meeting Quantitation strippedhuman urine. Inaccuracy and accuracy and precision criteria (LLOQ)Imprecision data was used. Response at LLOQ is ≥ 5 times the response ofzero calibrator Upper Limit of Prostaglandin D2 diluted in charcoalstripped Highest concentration meeting Quantitation human urine.Inaccuracy and Imprecision accuracy and precision criteria (ULOQ) datawas used. Blank Matrix Six lots of blank matrix were analyzed in Blankand zero calibrator are Effect triplicate free of interference at theretention times of analyte and Internal Standard Response of loweststandard is at least 5 times blank response Double blank response at theIS retention time is ≤ 5% of average IS of calibrators and QC in thesame run Internal Internal Standard in blank matrix was Blank with ISadded <LLOQ. Standard injected as sample. Interference LC system Thehigh standard (10000 pg/mL) followed Response of the blank followingcarry-over by a double blank was analyzed in four runs. a high sampleshould be less than evaluation the LLOQ. Spike and Human urine andcalibrator with low level Mean % Recovery from Recovery concentrationsof Prostaglandin D2 were expected (baseline concentration spiked withProstaglandin standard material plus spike) 85-115% (80-120% to low, midand high concentration. Baseline at LLOQ) and spiked samples were testedin triplicate. At least two-thirds of the sample replicates testedwithin 85-115% recovery. Dilution Diluted human urine (X2, X4, and X10).CV ≤ 15%; (LLOQ ≤ 20%) Linearity Three urine samples were analyzed neatand 85-115% of expected values (AMR diluted X2, X4, and X10. Fivereplicates for (based on measurement of neat, verification) eachdilution were analyzed for each urine undiluted urine) at each dilutionsample. level (80-120% if near LLOQ) Extraction Urine samples werespiked before and after No other criteria Recovery SPE processing atlow, medium and high concentrations. Recovery of samples spiked beforeSPE processing was compared to those spiked after SPE processing.Autosampler Autosampler stability was evaluated using Mean post-storagerecovery 85- Stability calibrators and quality control samples. 115%(80-120% at LLOQ) of the Duplicate sample sets were included with themean pre-storage concentration batch. The first sample set was injected,then with at least two-thirds of the after 3 days the entire batch wasinjected or sample replicates tested within re-injected. 85-115%recovery. Short-term Short-term sample stability was determined Mean %Recovery from baseline Stability by testing freshly collected humanurine 85-115% (80-120% at LLOQ), (spiked if necessary) that was storedunder with at least two-thirds of the conditions likely to beencountered in sample sample replicates at a particular handling andlaboratory analysis. condition tested within 85-115% One aliquot of eachsample was analyzed on recovery. the same day as preparation, and anadditional aliquot of each sample was placed into storage at ≤−55° C.The other aliquots were incubated at room temperature (15-30° C.),refrigerated (2-8° C.), and frozen (≤−10° C.) conditions, then placedinto storage at ≤−55° C. until analysis. All samples were analyzed intriplicate. Excluding those tested on the same day as preparation, allsamples for a given donor were analyzed in a single batch. Freeze/thawSample freeze/thaw stability was determined Mean % Recovery frombaseline Stability using aliquots of the collected human urine 85-115%(80-120% at LLOQ), used to evaluate short-term stability. One with atleast two-thirds of the set of aliquots at each level was analyzed onsample replicates at a particular the day of draw, another set wasstored condition tested within 85-115% at ≤−55° C., and the remainingset will be recovery. subjected to an additional 6 freeze/thaw cycles.

Inter- and intra-assay standard accuracy and precision. Five levels ofProstaglandin D2 spiked into charcoal stripped human urine were assayedover seven assay batches. All levels were analyzed twenty times in asingle batch and three times in 5 batches, with the exception ofaccuracy sample Level AA at 1 pg/mL. One validation batch(PGD2_VB1_052020) had low level contamination that was traced to theReconstitution Solution and therefore analysis of 20 replicates of the 1pg/mL accuracy sample was repeated for this batch. Accuracy at the lowend of the calibration curve was negatively impacted and accuracy andimprecision at 1 pg/mL was omitted from overall validation calculations.The experiment for analysis of 20 replicates of the accuracy sampleLevel AA at 1 pg/mL was repeated. A total of 175 individual results(20×5+15×5) were collected and analyzed. The samples were chosen to fallwithin different regions of the reportable range. Accuracy and precisionresults are summarized in TABLE 5 for the five concentrations rangingfrom 1-1000 pg/mL. Inter-assay and Intra-assay study results metacceptance criteria for accuracy and precision (TABLE 4).

TABLE 5 Inter- and Intra-Assay Standard Accuracy and Precision MethodValidation: Inaccuracy and Imprecision Component: Prostaglandin D2Sample Matrix: Charcoal Stripped Human Urine Accuracy SampleIdentification AA A1 A5 A6 A7 Target Concentration (pg/mL) 1.00 10.0 300500 1000 85% (80% at AA) of Target Concentration (pg/mL) 0.800 8.50 255425 850 115% (120% at AA) of Target Concentration (pg/mL) 1.20 11.5 345575 1150 Batch # Measured Concentration (pg/mL) PGD2_VB1_052020Intra-assay Mean 1.660 10.32 325.3 525.0 1028.7 Intra-assay StandardDeviation 0.662 0.49 8.1 18.7 71.6 Intra-assay Inaccuracy (% Bias) 66.03.2 8.4 5.0 2.9 Intra-assay Imprecision (% CV) 39.9 4.7 2.5 3.6 7.0 #Replicates within 85-115% (80-120% at LLOQ) of 4 3 3 3 3 TargetConcentration % Replicates within 85-115% (80-120% at LLOQ) 20.0% 100.0%100.0% 100.0% 100.0% of Target Concentration N 20 3 3 3 3PGD2_VB2_052120 Intra-assay Mean 1.160 8.88 290.0 524.3 1019.3Intra-assay Standard Deviation 0.157 0.52 15.4 12.9 69.9 Intra-assayInaccuracy (% Bias) 16.0 −11.3 −3.3 4.9 1.9 Intra-assay Imprecision (%CV) 13.5 5.9 5.3 2.5 6.9 # Replicates within 85-115% (80-120% at LLOQ)of 14 14 3 3 3 Target Concentration % Replicates within 85-115% (80-120%at LLOQ) 70.0% 70.0% 100.0% 100.0% 100.0% of Target Concentration N 2020 3 3 3 PGD2_VB3_052620 Intra-assay Mean 0.940 9.94 269.6 458.6 991.3Intra-assay Standard Deviation 0.178 0.48 12.1 18.2 11.6 Intra-assayInaccuracy (% Bias) −6.0 −0.6 −10.2 −8.3 −0.9 Intra-assay Imprecision (%CV) 18.9 4.9 4.5 4.0 1.2 # Replicates within 85-115% (80-120% at LLOQ)of 2 3 18 19 3 Target Concentration % Replicates within 85-115% (80-120%at LLOQ) 66.7% 100.0% 90.0% 95.0% 100.0% of Target Concentration N 3 320 20 3 PGD2_VB4_052720 Intra-assay Mean 1.161 9.70 324.0 567.7 1068.5Intra-assay Standard Deviation 0.263 0.49 20.2 32.5 33.3 Intra-assayInaccuracy (% Bias) 16.1 −3.0 8.0 13.5 6.8 Intra-assay Imprecision (%CV) 22.7 5.0 6.2 5.7 3.1 # Replicates within 85-115% (80-120% at LLOQ)of 2 3 3 2 20 Target Concentration % Replicates within 85-115% (80-120%at LLOQ) 66.7% 100.0% 100.0% 66.7% 100.0% of Target Concentration n 3 33 3 20 PGD2_VB5_052820 Intra-assay Mean 0.957 10.40 335.7 523.3 1034.0Intra-assay Standard Deviation 0.046 0.60 17.0 30.6 63.2 Intra-assayInaccuracy (% Bias) −4.3 4.0 11.9 4.7 3.4 Intra-assay Imprecision (% CV)4.8 5.7 5.1 5.8 6.1 # Replicates within 85-115% (80-120% at A1) of 3 3 23 3 Target Concentration 100.0% 100.0% 66.7% 100.0% 100.0% % Replicateswithin 85-115% (80-120% at A1) of Target Concentration n 3 3 3 3 3PGD2_VB6_060120 Intra-assay Mean 0.874 10.67 311.3 485.7 951.3Intra-assay Standard Deviation 0.060 0.74 16.9 31.1 41.8 Intra-assayInaccuracy (% Bias) −12.6 6.7 3.8 −2.9 −4.9 Intra-assay Imprecision (%CV) 6.8 6.9 5.4 6.4 4.4 # Replicates within 85-115% (80-120% at LLOQ) of3 3 3 3 3 Target Concentration % Replicates within 85-115% (80-120% atLLOQ) 100.0% 100.0% 100.0% 100.0% 100.0% of Target Concentration n 3 3 33 3 PGD2_VB8_060420 Intra-assay Mean 0.837 NA NA NA NA Intra-assayStandard Deviation 0.090 NA NA NA NA Intra-assay Inaccuracy (% Bias)−16.3 NA NA NA NA Intra-assay Imprecision (% CV) 10.7 NA NA NA NA #Replicates within 85-115% (80-120% at LLOQ) of 2 NA NA NA NA TargetConcentration % Replicates within 85-115% (80-120% at LLOQ) 66.7% NA NANA NA of Target Concentration n 3 NA NA NA NA Average Intra-assayInaccuracy (% Bias) 8.4 −0.1 3.1 2.8 1.6 Average Intra-assay Imprecision(% CV) 16.7 5.5 4.8 4.7 4.8 Inter-assay Mean 1.072 9.45 290.0 487.11041.2 Inter-assay Standard Deviation 0.192 0.86 29.0 42.5 54.6Inter-assay Inaccuracy (% Bias) 7.2 −5.5 −3.3 −2.6 4.1 Inter-assayImprecision (% CV) 17.9 9.1 10.0 8.7 5.2 n 35 35 35 35 35

Sample precision. 6 replicates of three levels of Prostaglandin D2spiked into human urine pools were assayed on 4 separate days. A totalof 24 individual results (6×4) were collected and analyzed for sampleprecision. Results are summarized in TABLE 6. Results showed that theacceptance criteria were met.

TABLE 6 Sample Precision Method Validation: Imprecision Component:Prostaglandin D2 Sample Matrix: Human Urine Normal Normal Normal Pool 1:Pool 2: Pool 3: UPG2 QC1, UPG2 QC 2, UPG3 QC3, Sample Identification Lot20142 Lot 20142 Lot 20141 Batch # Measured Concentration (pg/mL)PGD2_VB1_052020 Intra-assay Mean NA NA 804.5 Intra-assay StandardDeviation NA NA 27.6 Intra-assay Imprecision (% CV) NA NA 3.4 N NA NA 6PGD2_VB2_052120 Intra-assay Mean 6.64 118.8 728.3 Intra-assay StandardDeviation 0.87 4.1 16.4 Intra-assay Imprecision (% CV) 13.2 3.4 2.2 N 66 6 PGD2_VB3_052620 Intra-assay Mean 6.02 102.8 669.0 Intra-assayStandard Deviation 0.85 10.0 18.4 Intra-assay Imprecision (% CV) 14.29.7 2.8 N 6 6 6 PGD2_VB4_052720 Intra-assay Mean 5.95 107.2 724.0Intra-assay Standard Deviation 0.47 9.7 27.4 Intra-assay Imprecision (%CV) 7.9 9.1 3.8 N 6 6 6 PGD2_VB5_052820 Intra-assay Mean 6.40 101.2 NAIntra-assay Standard Deviation 1.02 8.0 NA Intra-assay Imprecision (%CV) 16.0 7.9 NA N 6 6 0 Average Intra-assay Imprecision 12.8 7.5 3.1 (%CV) Inter-assay Mean 6.25 107.51 731.46 Inter-assay Standard Deviation0.82 10.45 53.73 Inter-assay Imprecision (% CV) 13.2 9.7 7.3 N 24 24 24

Sensitivity: LLQQ and ULOQ. The Upper and Lower Limits of Quantitationwere determined using the materials used to show Intra- and Inter-AssayAccuracy and Imprecision. The Upper and Lower Limits of Quantitationwere determined from data collected during Intra- and Inter-AssayAccuracy and Imprecision Testing. The LLOQ is the lowest activity tomeet acceptance criteria and the ULOQ is the highest concentration tomeet acceptance criteria. The LLOQ could be demonstrated at 1 pg/mL andULOQ could be demonstrated at 1000 pg/mL.

Blank matrix Effect. To demonstrate blank matrix effect, six lots ofpotential blank matrix products were analyzed without the addition ofinternal standard (IS). Three replicates of each product were processedand analyzed to determine the effect of different lots of blank matrix.The blank and zero calibrator were free of interference at the retentiontimes of the analyte (PGD2) and the internal standard. Response of thelowest internal standard was at least five times the response of theblank. The double blank response at the IS retention time was ≤5% ofaverage IS of calibrators and QC in the same run.

Internal Standard Interference. To demonstrate internal standardinterference, a single lot of blank matrix product was analyzed with theaddition of internal standard. Three replicates of blank matrix wereprocessed and analyzed to determine the effect of internal standardinterference. When the IS in blank matrix was injected as the sample,the mean concentration was determined to be 0.00 pg/mL. InternalStandard added to blank matrix meets acceptance criteria (TABLE 4).

LC System carry-over. To demonstrate LC system Carry-Over, the doubleblank following each of the high standards in four assay batches wasanalyzed. The mean percent carry-over was 0.0%. Carry-over study resultsmet acceptance criteria (TABLE 4).

Spike and recovery. Human urine and charcoal stripped human urine withlow spiked levels of Prostaglandin D2 were additionally spiked withProstaglandin D2 standard material at low, mid, and high concentrations.Baseline (low spike) and additionally spiked urine were tested intriplicate. Percent recovery was based on mean baseline concentration oflow-spiked material plus the additional theoretical spiked concentration(TABLE 4). Results are summarized in TABLE 7.

TABLE 7 Spike and Recovery Method Validation: Spike and RecoveryComponent(s): Prostaglandin D2 Sample Matrix: Human Urine and CharcoalStripped Human Urine Validation Batch: PGD2_VB6_060120 andPGD2_VB9_060820 Concentration Added to Baseline (pg/mL) 0.0 25.0 250.01750.0 0.0 625.0 Measured Concentration (pg/mL) Human Urine 3.60

1780.0 5.60 666 (Normal Urine 1) 4.15 24.8 218.0 1660.0 5.45 613 2.1425.2 253.0 1570.0 5.07 610 Mean Conc. 3.30 24.6 227.7 1670.0 5.37 629.7Expected Conc. NA 28.3 253.3 1753.3 NA 630.4 Recovery (%) NA 87.1 89.995.2 NA 99.9 85% of Expected NA 24.1 215.3 1490.3 NA 535.8 Concentration115% of Expected NA 32.5 291.3 2016.3 NA 724.9 Concentration N 3 3 3 3 33 Charcoal Stripped Human 20.60 41.7 244.0 1630.0 18.7 649 Urine (A2)21.20 40.1 251.0 1560.0 16.4 621 21.00 42.9 257.0 1750.0 19.4 584 Mean20.93 41.6 250.7 1646.7 18.17 618.0 Expected Conc. NA 45.9 270.9 1770.9NA 643.2 Recovery (%) NA 90.5 92.5 93.0 NA 96.1 85% of Expected NA 39.0230.3 1505.3 NA 546.7 Concentration 115% of Expected NA 52.8 311.62036.6 NA 739.6 Concentration N 3 3 3 3 3 3 Note: Expected concentrationis equal to the mean baseline concentration plus the concentration addedto baseline. Note: Samples listed in

 were not within 85-115% of expected concentration. Note: Original highspike of 1750 pg/mL was initially performed at a level outside the ULOQof the assay platform (1000 pg/mL). Data was included and high spikeexperiment was repeated at a level within the assay limits.

Dilutional linearity (AMR verification). To demonstrate linearity ofdilution, three human urine samples were assayed at reduced volume. Thefinal dilution factors for the samples analyzed were X1 (neat, normalvolume), X2, X4 and X10. Each sample and volume was tested five times inone assay. Expected values were calculated based on averageconcentrations of the samples run at normal volume (400 uL). Results aresummarized in TABLE 8. Dilutional Linearity study results met acceptancecriteria for urine samples analyzed using the alternative volumes of 200uL and 100 uL in addition to the standard sample volume for the assay(400 uL) (TABLE 4). Samples analyzed using 40 uL of urine passed for ⅔samples tested and failed for the third sample. 40 uL of urine is notacceptable for analysis.

TABLE 8 Dilutional Linearity Method Validation: Linearity (AMRVerification) Component(s): Prostaglandin D2 Sample Matrix: Human UrineValidation Batch: PGD2_VB9_060820 Sample Volume (uL) 400 200 100 40Dilution Factor Neat X2 X4 X10 Calculated Concentration SampleIdentification (pg/mL) Dilution Sample 1 46.4  53.6  55.9  57.6 52.3 48.8  57.3  49.2 48.0  55.4  49.2   

51.0  52.2  53.9   

51.3  53.7  53.7  51.8 Mean 49.8  52.7  54.0  57.8 Imprecision (% CV) 5.0%  4.7%  5.7%  15.2% Mean % Recovery Compared NA 105.9% 108.4%116.1% to Neat 85% of Neat Concentration 42.3 NA NA NA 115% of NeatConcentration 57.3 NA NA NA Dilution Sample 2 66.9  68.9  72.6   

73.9  74.1  78.3  64.3 75.7  74.9  82.8  78.0 76.9  71.9   

 79.8 74.5  81.0  77.5  72.1 Mean 75.3  74.2  74.9  71.0 Imprecision (%CV)  5.2%  6.0%  10.0%  11.7% Mean % Recovery Compared NA  98.6%  99.5% 94.4% to Neat 85% of Neat Concentration 64.0 NA NA NA 115% of NeatConcentration 86.5 NA NA NA Dilution Sample 3 75.7  73.8  73.2  80.180.2  87.1  83.5  74.9 84.7  87.5  88.5  75.2 82.5  74.3  81.6  83.678.2  77.1  85.3  83.1 Mean 81.4  80.0  82.4  79.4 Imprecision (% CV) 4.3%  8.5%  7.0%  5.3% Mean % Recovery Compared NA  98.2% 101.3%  97.5%to Neat 85% of Neat Concentration 69.2 NA NA NA 115% of NeatConcentration 93.6 NA NA NA Mean Imprecision (% CV)  4.8%  6.4%  7.5% 10.7% Total replicates within 85- NA 100.0%  93.3%  80.0% 115% of NeatOverall Mean % Recovery NA 100.9% 103.1% 102.7% Compared to Neat Note:Samples listed in  

  were not within 85-115% of neat calculated concentration.

Extraction recovery. To demonstrate extraction recovery, human urine andcharcoal stripped human urine with low spiked levels of Prostaglandin D2were additionally spiked with Prostaglandin D2 standard material at low,mid, and high concentrations both before and after SPE plate processing.Each sample was tested in triplicate. Expected values were calculatedbased on average concentrations found when spiking samples before SPEplate processing. Results are summarized in TABLE 9.

TABLE 9 Extraction Recovery Method Validation: Extraction RecoveryComponent(s): Prostaglandin D2 Sample Matrix: Human Urine ValidationBatch: PGD2_VB6_060120¹ and PGD2_VB9_060820² Pre- or Post-ExtractionSpike Pre-Spiked Post-Spiked Sample Identification CalculatedConcentration (pg/mL) Low Cal/Acc_Low Spike¹  41.7  32.1  40.1  35.4 42.9  39.4 Mean  41.6  35.6 Mean % Recovery Compared to NA  85.7%Pre-Spike 85% of Pre-Spike Concentration  35.3 NA 115% of Pre-SpikeConcentration  47.8 NA Low Cal/Acc_Mid Spike¹  244  279  251  310  257 266 Mean  250.7  285.0 Mean % Recovery Compared to NA  113.7% Pre-Spike85% of Pre-Spike Concentration  213.1 NA 115% of Pre-Spike Concentration 288 NA Low Cal/Acc_High Spike²  649  668  621  686  584  651 Mean 618.0  668.3 Mean % Recovery Compared to NA  108.1% Pre-Spike 85% ofPre-Spike Concentration  525 NA 115% of Pre-Spike Concentration  711 NA*Low Cal/Acc_High Spike¹ 1630* 1830* 1560*  

1750*  

Mean 1646.7 1910.0 Mean % Recovery Compared to NA  116.0% Pre-Spike 85%of Pre-Spike Concentration 1400 NA 115% of Pre-Spike Concentration 1894NA Low Sample_Low Spike¹  23.9  21.4  24.8  24.0  25.2   

Mean  24.6  21.4 Mean % Recovery Compared to NA  86.7% Pre-Spike 85% ofPre-Spike Concentration  20.9 NA 115% of Pre-Spike Concentration  28.3NA Low Sample_Mid Spike¹  212   

 218   

 253   

Mean  227.7  275.7 Mean % Recovery Compared to NA  121.1% Pre-Spike 85%of Pre-Spike Concentration  194 NA 115% of Pre-Spike Concentration  262NA Low Sample_High Spike²  666  687  613  657  610  695 Mean  629.7 679.7 Mean % Recovery Compared to NA  107.9% Pre-Spike 85% of Pre-SpikeConcentration  535 NA 115% of Pre-Spike Concentration  724 NA *LowSample_High Spike¹ 1780* 1900* 1660* 1920* 1570* 1820* Mean 1670.01880.0 Mean % Recovery Compared to NA  112.6% Pre-Spike 85% of Pre-SpikeConcentration 1420 NA 115% of Pre-Spike Concentration 1921 NA Note:Samples listed in  

  were not within 85-115% of neat calculated concentration. Note:*Original high pre- and post-spike of 1750 pg/mL was accidentallyperformed at a level outside the ULOQ of the assay platform (1000pg/mL). Data was included and pre- and post-high spike experiment wasrepeated at a level within the assay limits.

Autosampler stability. To demonstrate autosampler stability qualitycontrol samples, calibrators, and normal urine samples were analyzed. Tovalidate autosampler stability a batch containing two sets ofcalibrators and QCs was processed as normal. After completion of assayprocessing the first set of calibrators, QCs, and normal urines wasinjected and analyzed. After analysis the assay batch was storedrefrigerated in the autosampler prior to reinjection of the first set ofsamples and first time injection of the second set of calibrators andQCs. The 96-well plates containing the processed assay were storedrefrigerated in the autosampler for approximately 3 days, 8 hours, and36 minutes before injection was completed for all samples. Post-storagerecoveries were based on target concentrations for the stored, firsttime injection samples. Reinjected samples were compared to initialinjection results to determine recoveries. Autosampler study results metacceptance criteria. Mean post-storage recovery with 85-115% (80-120% atLLOQ) of the mean pre-storage concentration with at least two-thirds ofthe sample replicates tested within 85-115% recovery (TABLE 4). Assaybatches stored refrigerated for up to 3 days and 8 hours were stable andsuitable for first time injection or re-injection to determineProstaglandin D2 concentrations in human urine.

Reference interval. To establish a reference interval one hundred andtwenty-two human urine samples were evaluated. The individual urinesamples were collected in-house or purchased from GoldenWestBioSolutions and the individuals tested did not have a known history ofrelated disease or condition. The reference range samples were analyzedin six of the validation batches (PGD2_VB4_052720, PGD2_VB5_052820,PGD2_VB6_060120, PGD2_VB7_060320, PGD2_VB8_060420, and PGD2_VB9_060820).Six of the normal samples (Normal Sample IDs 11, 14, 30-31, and 45-46)had to be analyzed at reduced volume because there was limited samplevolume available. Two of these normal samples, Normal Sample IDs 30 and31, both produced results that were below the limit of quantitation forthe assay platform (1 pg/mL) and were therefore not included in the dataanalysis for reference interval establishment. The other normal samplesthat produced results within the measurable range of the assay when runon reduced volume were included in the data analysis for referenceinterval establishment. Two of the normal samples (Normal Sample IDs 16and 45) were flagged by the EP Evaluator Software as outliers and werenot included in the data to establish the reference interval. Theresults for Prostaglandin D2 concentration in human urine werenormalized to each patient's urine creatinine level in order toestablish a clinical reference range. The following equation was used tonormalize the Prostaglandin D2 levels to urine creatinine levels:

$\begin{matrix}{{Normalized}\mspace{14mu}{Prostaglandin}\mspace{14mu}{D2}} \\{{Concentration}\mspace{14mu}\left( {{ng}\mspace{14mu}{PGD}\; 2\text{/}g\mspace{14mu}{creatinine}} \right)}\end{matrix}\frac{\begin{matrix}{{Urine}\mspace{11mu}{Prostaglandin}\mspace{14mu}{D2}} \\{{Concentration}\mspace{14mu}\left( {{pg}\text{/}{mL}} \right) \times 100}\end{matrix}\;}{{Urine}\mspace{14mu}{Creatinine}\mspace{14mu}{Level}\mspace{14mu}\left( {{mg}\text{/}{dL}} \right)}$

Results are summarized in Table 10. The reference interval forNormalized Prostaglandin D2 Concentration in urine as determined by the97.5th percentile will be <52.6 ng PGD2/g creatinine.

TABLE 10 Reference Interval Method Validation: Reference IntervalComponent(s): Prostaglandin D2 Sample Matrix: Human Urine SamplesPGD2_VB4_052720, PGD2_VB5_052820, PGD2_VB6_060120, PGD2_VB7_060320,PGD2_VB8_060420, and PGD2_VB9_060820 Normalized Urine PGD2 MeasuredUrine Concentration Concentration Creatinine (ng PGD2/g Sample ID(pg/mL) (mg/dL) creatinine) 1 7.18 161.69 4.44 2 6.30 53.60 11.8 3 2.6637.63 7.07 4 6.52 144.97 4.50 5 31.3 74.66 41.9 6 4.61 198.59 2.32 711.1 262.57 4.23 8 5.03 16.91 29.7 9 10.7 23.10 46.3 10 71.8 81.54 88.1

12 29.7 117.30 25.3 13 17.2 63.91 26.9

15 1.52 219.12 0.694 16 31.5 21.80 144 17 14.6 200.83 7.27 18 21.1145.89 14.5 19 14.8 127.84 11.6 20 16.7 200.62 8.32 21 7.87 63.45 12.422 4.63 279.11 1.66 23 13.6 188.35 7.22 24 23.0 86.26 26.7 25 13.3 91.9214.5 26 30.7 134.92 22.8 27 10.6 101.98 10.4 28 12.3 199.50 6.17 29 53.5111.02 48.2

32 25.6 163.53 15.7 33 17.9 320.55 5.58 34 10.4 149.09 6.98 35 12.558.04 21.5 36 2.79 58.45 4.77 37 23.2 110.10 21.1 38 31.5 143.24 22.0 399.04 307.37 2.94 40 13.2 82.09 16.1 41 5.38 149.10 3.61 42 14.2 257.335.52 43 1.01 124.76 0.810 44 7.64 231.16 3.31

47 5.97 364.04 1.64 48 9.48 25.63 37.0 49 9.40 35.93 26.2 50 24.8 151.2616.4 51 2.46 178.26 1.38 52 21.7 223.89 9.69 53 12.2 109.19 11.2 54 7.25183.58 3.95 55 4.35 179.61 2.42 56 9.19 252.66 3.64 57 1.95 225.20 0.86658 5.72 44.27 12.9 59 4.58 118.17 3.88 60 25.4 292.16 8.69 61 23.6301.91 7.82 62 26.9 184.47 14.6 63 10.3 124.24 8.29 64 21.7 103.21 21.065 1.79 130.49 1.37 66 8.92 49.33 18.1 67 8.09 141.31 5.73 68 4.33 95.664.53 69 3.19 247.74 1.29 70 0.00 220.56 0.000 71 8.77 182.55 4.80 7210.10 324.09 3.12 73 1.91 291.94 0.654 74 10.7 175.26 6.11 75 7.25373.40 1.94 76 4.52 291.15 1.55 77 9.28 18.25 50.8 78 2.58 213.32 1.2179 8.98 63.48 14.1 80 21.1 141.36 14.9 81 5.72 131.08 4.36 82 12.2260.22 4.69 83 1.65 275.83 0.598 84 44.6 114.38 39.0 85 16.8 200.13 8.3986 21.7 41.04 52.9 87 3.48 282.78 1.23 88 20.7 126.47 16.4 89 10.4252.59 4.12 90 23.3 74.48 31.3 91 19.1 204.41 9.34 92 12.7 161.75 7.8593 20.1 126.90 15.8 94 33.6 324.66 10.3 95 8.76 268.35 3.26 96 24.5176.87 13.9 97 0.00 167.28 0.000 98 9.42 210.13 4.48 99 11.9 116.99 10.2100 15.0 195.90 7.66 101 21.1 297.99 7.08 102 19.2 237.02 8.10 103 13.4230.51 5.81 104 6.87 318.93 2.15 105 38.9 86.23 45.1 106 18.9 188.8010.0 107 48.7 156.59 31.1 108 10.7 33.16 32.3 109 0.00 209.67 0.000 1105.15 151.98 3.39 111 6.75 160.22 4.21 112 30.1 184.70 16.3 113 31.9221.70 14.4 114 23.1 120.39 19.2 115 15.8 207.79 7.60 116 30.8 50.3061.2 117 4.26 230.64 1.85 118 16.3 136.44 11.9 119 13.4 109.57 12.2 1203.98 126.04 3.16 121 1.34 193.23 0.693 122 5.97 83.13 7.18 Mean 16.122165.545 14.726 Concentration (pg/mL) Standard 22.355 83.945 20.876Deviation

Selectivity. To demonstrate selectivity, potential interferents in thepresence of Prostaglandin D2 were analyzed. Multiple replicates of anaccuracy sample were processed as normal and the interferents were addedindividually to the 12×75 glass tubes prior to sample addition andpre-SPE processing. Baseline samples (with no added interferent) wereanalyzed in triplicate and samples containing interferents were analyzedin singlicate. Recovery in the presence of interferents was based on themean concentration of the baseline sample. Results are summarized inTABLE 11. Not all potential interferents passed selectivity studyacceptance criteria. Δ12-Prostaglandin D2 and 15(R)-Prostaglandin D2had >85-115% recovery from baseline and therefore did not meetacceptance criteria (TABLE 4). Δ12-Prostaglandin D2 is one of theinitial chemical decomposition products of PGD2. The retention time ofΔ12-Prostaglandin D2 is 1.14 minutes with the retention time ofProstaglandin D2 being in the range of 1.58-1.71 minutes (Refer to TABLE12 for retention times). The measured concentration in TABLE 12 is mostlikely due to a result of chemical impurities in the commerciallyavailable product. While 15(R)-Prostaglandin D2 is commerciallyavailable it has not been reported at as an endogenous metabolite ofProstaglandin D2. It will therefore not cause interference issues withthe assay platform when analyzing human urine.

TABLE 11 Selectivity Method Validation: Selectivity Component(s):Prostaglandin D2 with Potential Interferents Sample Matrix: CharcoalStripped Human Urine spiked with Prostaglandin D2 and PotentialInterferents Validation Batch: PGD2_VB9_060820 and PGD2_VB10_061520Measured Mean Measured Sample Identification Concentration (pg/mL)Concentration (pg/mL) No Interferent- 18.7 18.2 Sample Baseline 16.419.4 Interferent Measured % Recovery from Identification Concentration(pg/mL) Sample Baseline Specificity 1  17.5  96.3% (2,3-dinor-11b-Prostaglandin F2a) Specificity 2  15.5  85.3% (13,14-dihydro-15-ketoProstaglandin D2) Specificity 3  18.9 104.0% (PGDM) Specificity 4  17.8 98.0% (Δ2-Prostaglandin J2) Specificity 5  16.4  90.3% (tetranor-PGJM)Specificity 6  19.6 107.9% (11b-13,14-dihydro-15- keto ProstaglandinF2a) Specificity 7  19.1 105.1% (5-trans Prostaglandin D2) Specificity8  19.0 104.6% (Prostaglandin F2a) No Interferent- 18.7 18.2 SampleBaseline 16.4 19.4 Specificity 9  16.3  89.7% (8-isoProstaglandin F2a)Specificity 10 15.7  86.4% (15-deoxy-Δ12,14- Prostaglandin D2)Specificity 11 164 902.8% (Δ12-Prostaglandin D2) Specificity 12 18.5101.8% (ent-Prostaglandin F2a) Specificity 13 653 3594.5% (15(R)-Prostaglandin D2) No Interferent- 15.7 15.7 Sample Baseline 16.814.6 Specificity 14 15.1  96.2% (Prostaglandin E2)

Specificity. To demonstrate specificity, blank matrix spiked withpotential interferents were analyzed. All samples were analyzed insinglicate. Not all potential interferents passed specificity studyacceptance criteria. Δ12-Prostaglandin D2 recovered at 14.7% and15(R)-Prostaglandin D2 recovered at 31.3% did not meet acceptancecriteria (TABLE 4). Δ12-Prostaglandin D2 is one of the initial chemicaldecomposition products of PGD2. The retention time of Δ12-ProstaglandinD2 is 1.14 minutes with the retention time of Prostaglandin D2 being inthe range of 1.58-1.71 minutes. While 15(R)-Prostaglandin D2 iscommercially available it has not been reported at as an endogenousmetabolite of Prostaglandin D2. It will therefore not cause interferenceissues with the assay platform when analyzing human urine.

TABLE 12 Specificity Method Validation: Specificity Component(s):Prostaglandin D2 Sample Matrix: Charcoal Stripped Human Urine Spikedwith Potential Interferents Assay Date: PGD2_VB9_060820 andPGD2_VB10_061520 Spiked Retention Measured Molecular Concentration TimeConcentration % Sample ID Interferent Compound Weight (pg/mL) (Minutes)(pg/mL) Recovery Specificity 1 2,3-dinor-11β-Prostaglandin F2α 326.41000 Not in 0.176 0.0% Window Specificity 2 13,14-dihydro-15-keto 352.51000 Not in 0.136 0.0% Prostaglandin D2 Window Specificity 3 PGDM 328.41000 Not in 0.228 0.0% Window Specificity 4 Δ12-Prostaglandin J2 334.51000 1.04 0.191 0.0% Specificity 5 tetranor-PGJM 310.3 1000 Not in 0.2280.0% Window Specificity 6 11β-13,14-dihydro-15-keto 354.5 1000 Not in0.191 0.0% Prostaglandin F2α Window Specificity 7 5-trans ProstaglandinD2 352.5 1000 1.40 25.2 2.5% Specificity 8 Prostaglandin F2α 354.5 1000Not in 0.541 0.1% Window Specificity 9 8-isoProstaglandin F2α 354.5 1000Not in 0.227 0.0% Window Specificity 10 15-deoxy-Δ12,14-Prostaglandin D2316.4 1000 Not in 0.348 0.0% Window Specificity 11 Δ12-Prostaglandin D2352.5 1000 1.14 147 14.7% Specificity 12 ent-Prostaglandin F2α 354.51000 Not in 0.495 0.0% Window Specificity 13 15(R)-Prostaglandin D2352.5 1000 1.66 313 31.3% Specificity 14 Prostaglandin E2 352.5 352.50.72 No Peak 0.0% Retention time of Prostaglandin D2 ranged from1.58-1.71 minutes for batches that Selectivity Samples were run in.

Calibration or standard curve accuracy and precision. Eleven standardpoints (ten of which were non-zero) were included in each run to definethe calibration curve. A standard curve was generated using tenstandards having concentrations of 0, 1, 2, 5, 10, 30, 50, 100, 300, 500and 1000 pg/mL. The lowest non-zero point has a target concentration of1 pg/mL Prostaglandin D2. Analyst software was used to plot the datausing a quadratic fit function with 1/x weighting. Standard curveback-fit data from all ten validation batches was tabulated (with 13total standard curves tabulated—the autosampler stability batch had morethan one set of standards and one of the standard curves was re-injectedfor autosampler reinjection stability). The correlation coefficient wasgreater than 0.99 for all replicates. Calibrator/Standard Curve resultsmet acceptation criteria for bias and precision.

Example 3. Measurement of PGD2 in Serum by SPE-LC-MS/MS

Specimens. All studies were performed using serum samples. Acceptablesample volumes are shown in TABLE 13.

TABLE 13 Specimen Collection Adult:   5 mL serum is acceptablePediatric: 2.5 mL serum is acceptable Minimum:   1 mL serum isacceptable

Serum samples were collected into red top tubes and frozen immediately.Specimens were stored according to the following conditions:

-   -   Short Term Storage        -   Serum Frozen (≤−10° C. and ≤−55° C.): 7 days        -   Serum Refrigerated (2-8° C.): Strictly frozen storage. Do            not store refrigerated.        -   Serum Room Temperature (15-30° C.): Strictly frozen storage.            Do not store at room temperature.        -   Serum Freeze/Thaw (≤−10° C.): 2 Cycles (three total thaws)    -   Long Term Storage        -   Serum Frozen (≤−10° C.): 70 days    -   Shipping Conditions:        -   Serum Frozen (≤−10° C.): on dry ice

Assay parameters. The assay was conducted according to the parametersprovided in TABLE 14.

TABLE 14 Assay Parameters Parameter Value or Criteria Reportable AMRMaximum Range (LLOQ-ULOQ) Dilution/Concentration Units of MeasureProstaglandin D2 1-1000 pg/mL X5/5000 pg/mL pg/mL Alert/Critical ValuesNot Applicable. Repeat Rules Repeat at reduced volume if greater than1000 pg/mL. Max Dilution Reduced volume down to 100 μL, is allowable.Preferred Specimen Serum collected in a red top tube is the onlyacceptable sample type.

Acceptance Criteria. No more than 25% of all standard curve values maybe deleted. Previous standard curve data may be used to identifystandard points to delete. Standard curve regression was based uponindividual replicates, and each replicate was considered a point foracceptance rules.

Reduced volume analysis. Undiluted samples with results above 1000 pg/mLwere retested at reduced volume. Samples can be analyzed using as littleas 100 μL when necessary. Dilution factor must be applied.

Assay procedure—sample preparation. Standards, controls, and sampleswere thawed and then vortexed three times. 500 μL of standards,controls, and samples were pipetted into the appropriately labeled 12×75glass tubes. Four double blank tubes were left empty. Using a repeaterpipette, 100 μL of internal standard was added to each tube, except forthe double blanks. The vials were then covered with parafilm andvortexed using a multi-tube vortex mixer 10 times. Next, 1.4 mL 1%formic acid in 10/90 acetonitrile/water was added to each tube and thenthe tubes were covered and vortexed for 10 minutes at 1200-1500 RPM.Tubes were then centrifuges at 3500 RPM for 10 minutes.

Assay procedure SPE plate processing. A Strata X Pro 96-well plate(Phenomenex Catalog #8E-5536-TGA, or equivalent) was placed on top of awaste collection plate. 1 mL of methanol was added to each well and thewells were drained by gravity without the use of positive pressure. 1 mLof 5% methanol in water was then added to each of the wells drained bygravity without the use of positive pressure. The entire volume ofdiluted standards, controls, and samples were added to the correspondingwells of the plate. The wells were first drained by gravity and thenpositive pressure was applied on low setting. 1 mL of 5% methanol inwater was added to each of the wells of the 96-well plate and the wellswere drained by gravity and then positive pressure was applied on lowsetting. 1 mL of 20% methanol in water with 2% ammonium hydroxide wasthen added to each of the wells and the wells drained by gravity andthen positive pressure was applied on low setting. Next, 1 mL of 5%methanol in water was added to each of the wells and after the wellswere drained by gravity positive pressure was applied on low setting.Next, 1 mL of 30% methanol in water with 2% formic acid was added toeach of the wells and after the wells were drained by gravity, positivepressure was applied on low setting. 1 mL of 5% methanol in water wasthen added to each of the wells and the wells drained by gravityfollowed by positive pressure applied on low setting. Next, 1 mL of 40%methanol in water was added to each of the wells and the wells weredrained by gravity and then positive pressure was applied on lowsetting. The positive pressure setting was switched to high for 1minute. Next, the plate was placed on top of a new 1.2 mL 96-wellcollection plate and 300 μL of 2% acetic acid in chloroform was added toeach of the wells of the sample/standard plate. The wells were drainedby gravity and then positive pressure was applied on low setting. Again,300 μL of 2% acetic acid in chloroform was added to each of the wells.The wells were again drained by gravity, and then positive pressure wasapplied on low setting. The 1.2 mL 96-well plate with collected sampleswas then placed into a TurboVap 96 Concentration Workstation (BiotageLife Sciences) for approximately 30 mins at 40° C. until samples weredry. Then, 120 μL of PGD₂ reconstitution solution (3.33 ng/mL PGD₂-d₄internal standard in 1:3 methanol:10 mM ammonium acetate) was added toall wells, the plate was sealed, and mixed four times for 30 seconds(for a total of 2 minutes). The 96-well plate was then positioned in theLC-MS/MS Autosampler.

HTLC-MS/MS Procedure. For HTLC purification of PGD₂, a PhenomenexKinetex 2.6 μm C18(2) 100 Å, 150×4.6 mm column was used. All LC systemreagents were filled and LC pumps were primed to remove any bubbles frommobile phase lines or to remove mobile phase from previous assays. Themass spectrometer was then equilibrated for 1 minute. The Aria system(Aria OS Version 1.4 or greater, Cohesive Technologies (MA, USA)) wasstarted. Aria TX4 HTLC System, Cohesive Technologies, (MA, USA)consisting of 4 each: 1100 Series Quaternary Pump, 1100 Series BinaryPump, 1100 Series Vacuum Degasser, or 8 Series 1200 Binary Pump and 4Series 1200 Vacuum Degasser were started and primed at 5 mL per minutefor 5 minutes for each solvent to be used. Test injections wereperformed using UPGD₂ system suitability test (SST). The loading pumpwas run using a gradient starting with 60% mobile phase A (0.1% formicacid in water) and 40% mobile phase B (100% acetonitrile) at a flow rateof 0.8 mL/min. The eluting pump was run with 100% mobile phase A and 0%mobile phase B at a flow rate of 0.00 mL/min.

For MS/MS, an AB SCIEX API5000 triple quadrupole mass spectrometer,operating in negative ion electrospray ionization (ESI) mode(Turboionspray) was used for detection. Quantification of analyte andinternal standard was performed in selected reaction monitoring mode(SRM) with the use of ion summing. For PGD₂ the 351.3→233.1 transitionwas monitored. For the internal standard (PGD₂-d₉), the 360.4→232.9transition was monitored. A resulting MS/MS scan of the PGD₂ systemsuitability test (SST) is shown in FIG. 5. For FIG. 5, PGD₂-H₂O peaks of351.300 and 333.100 and PGD₂ SST peaks of 351.298 and 233.100 wereanalyzed. The signal:noise (S/N) ratio was 5.5. The peak intensity was7.7e+2 counts per second (cps) with a Ymax of 2.2e+2 cps and Ymin of8.0e+1 cps.

For detection of multiple product ions from one or more precursor ionsthe mass spectrometer was operated in multiple reaction monitoring (MRM)mode. The following transitions were monitored for each of the analyteslisted below:

-   -   PGD2-H2O: 351.300→333.100    -   d9-PGD2-H2O: 360.400→342.200    -   d4-PGD2-H2O: 355.400→337.000    -   PGD2-2H2O: 351.300→315.300    -   d9-PGD2-2H2O: 360.400→324.400    -   d4-PGD2-2H2O: 355.400→319.200    -   PGD2: 351.3→271.2; 351.298→271.200; 351.299→271.200;        351.301→271.200; 351.302→271.200; 351.299→233.100;        351.298→233.1; 351.301→233.100; 351.302→233.100;        351.300→189.100; 351.298→189.100; 351.299→189.100;        351.301→189.100; 351.302→189.100; 351.300→251.200;        351.298→251.200; 351.299→251.200; 351.301→251.200;        351.302→251.200    -   d9-PGD2: 360.400→280.100; 360.400→232.900; 360.400→189.000;        360.400→250.900    -   d4-PGD2: 355.400→275.300; 355.400→237.300; 355.400→193.201;        355.400→255.500

Calculations. Integration parameters were set using the QuantitationWizard in the Analyst Version 1.4 or greater. Sciex, (CA, USA)) program.A standard curve was generated and a metric plot was generated withindex on the x-axis vs. the internal standard on the y-axis. A standardcurve was generated using ten standards having concentrations of 0, 1,2, 5, 10, 30, 50, 100, 300, 500 and 1000 pg/mL. Standard curves wereused to determine the amount of PGD₂ present in each sample. Duplicatecalibration curves were used for each batch of samples. A total of 25%of standard points may be excluded from the combined curves if theback-calculated concentrations exceed the theoretical concentrationsby >20% at the LLOQ or >15% at other concentrations. No result wasreported below the lowest, or above the highest remaining standard.Samples with values less than the minimum reportable dose werecalculated and reported as “less than” value. All chromatographic peakshapes were reviewed for consistency. Observation of peak distortionindicated the presence of a contaminant. Where multiple peaks wereobserved within the chromatogram, to ensure the correct peak wasintegrated, the retention of the peak integrated was confirmed tocorrespond to calibrators and quality control samples. Internal standardpeak areas vs. index plot were visually reviewed for gross indicationsof processing and/or technical errors. All internal standard peak areasthat visually appeared to be >50% of the neighboring peaks wereconsidered for repeat analysis. All internal standard peak areas thatvisually appeared to be 33% less than the neighboring peaks wereconsidered for repeat analysis. Such anomalies are good indicators ofreagent addition or pipetting errors or technical malfunctions ofequipment. The correlation coefficient was greater than 0.99 for allreplicates.

Example 4. Validation of the Measurement of PGD2 in Serum bySPE-LC-MS/MS

Standard Material. The standard lots were prepared by diluting thematerial from two separate vials of commercially available ProstaglandinD2 MaxSpec Standard purchased from Cayman Chemical Company into 6% BSAwith 3.6 μg/mL Indomethacin and 50 μg/mL BHT added for stability. Thecalibration standards used for validation ranged in concentration from1-1000 pg/mL.

Controls. Clinical Quality Control pools used during this validationwere prepared by diluting commercially available Prostaglandin D2MaxSpec Standard purchased from Cayman Chemical Company into human serumpools. Four control pools were used in each validation batch.

Test procedures. The assay steps were performed according to SerumProstaglandin D2 by SPE and LCMS, as described in Example 3. Thevalidation was completed in 10 independent assays (one of which wasanalyzed twice for establishment of autosampler stability).

Acceptance Criteria. The acceptance criteria for each of the followingvalidation assays are shown in TABLE 15.

TABLE 15 Acceptance Criteria PARAMETER MATERIAL ACCEPTANCE CRITERIAIntra-assay Five levels of Prostaglandin D2 diluted in Runs include anLLOQ Standard 6% BSA at LLOQ, low, mid, high, and calibrator levelAccuracy ULOQ target concentrations. Preparation of Exempt from QC andaccuracy and precision samples was acceptance criteria Precisionindependent of standard preparation. Bias ≤ ± 15%; (LLOQ 20%) Twentyreplicates of each level were CV ≤15%; (LLOQ ≤20%) analyzed in a singlebatch. Inter-assay The above samples were analyzed in six Runs includean LLOQ Standard batches, with a minimum of three replicates calibratorlevel Accuracy at each level. Batch processing was Exempt from QC andperformed using different acceptance criteria Precision reagent lots asavailable. Mean inter-assay bias ≤ ± 15%; (LLOQ 20%) and at least 2/3 ofintra-assay bias values ≤ ± 15% CV ≤15%; (LLOQ ≤20%) and at least 2/3 ofintra-assay CV values within range Intra-assay Human serum with high,medium and low CV ≤15%; (LLOQ ≤20%) Sample concentrations ofProstaglandin D2. Six Precision replicates of each level was analyzed ina single batch. Inter-assay The above samples were analyzed three CV≤15%; (LLOQ ≤20%) and Sample times on different days with different lotsof at least 2/3 of intra-assay Precision reagent if possible. CV valueswithin range Lower Prostaglandin D2 diluted in 6% BSA. Lowestconcentration meeting Limit of Inaccuracy and Imprecision data was used.accuracy and precision criteria Quantitation Response at LLOQ is ≥5times (LLOQ) the response of zero calibrator Upper Prostaglandin D2diluted in 6% BSA. Highest concentration meeting Limit of Inaccuracy andImprecision data was used. accuracy and precision criteria Quantitation(ULOQ) Blank Six lots of blank matrix Blank and zero calibrator are freeMatrix were analyzed in triplicate of interference at the retentiontimes Effect of analyte and Internal Standard Response of loweststandard is at least 5 times blank response Double blank response at theIS retention time is ≤5% of average IS of calibrators and QC in the samerun Internal Internal Standard in blank matrix Blank with IS added <LLOQ. Standard was injected as sample. Interference Effect of, Icteric,and hemolyzed samples were Recovery from baseline is 85- Icteric andprepared by adding using the Sun 115% in at least 2/3 of the samplesHemolyzed Assurance ® Interference Kit as directed tested at eachcondition. samples by the manufacturer. A minimum of 3 replicates weretested for each sample type. LC system The high standard (10000 pg/mL)followed Response of the blank following a carry-over by a double blankwas analyzed in four runs, high sample should be evaluation less thanthe LLOQ. Spike and Human serum and calibrator with low level Mean %Recovery from expected Recovery concentrations of Prostaglandin D2 were(baseline concentration plus spike) spiked with Prostaglandin standardmaterial 85-115% (80-120% at LLOQ) to low, mid and high concentration.Baseline At least two-thirds of the sample and spiked samples weretested in triplicate, replicates tested within 85-115% recovery.Dilution Reduced volumes of human serum (250 uL, CV ≤15%; (LLOQ ≤20%)Linearity 100 uL, and 50 uL) were analyzed. Three 85-115% of expectedvalues (based (AMR serum samples were analyzed using the on measurementof neat, undiluted verification) standard sample volume of 500 uL aswell as serum) at each dilution level the listed reduced volumes. Fivereplicates (80-120% if near LLOQ) for each sample volume were analyzedfor each serum sample. Dilution factors were applied. Extraction Serumsamples were spiked before and after No other criteria Recovery SPEprocessing at low, medium and high concentrations. Recovery of samplesspiked before SPE processing was compared to those spiked after SPEprocessing (TABLE 20). Autosampler Autosampler stability was evaluatedusing Mean post-storage recovery 85- Stability calibrators and qualitycontrol samples. 115% (80-120% at LLOQ) of the Duplicate sample setswere included with the mean pre-storage concentration with batch. Thefirst sample set was at least two-thirds of the sample injected, thenafter 3 days the entire replicates tested within batch was injected orre-injected. 85-115% recovery. Short-term Short-term sample stabilitywas determined Mean % Recovery from Stability by testing freshlycollected human serum baseline 85-115% (spiked if necessary) that wasstored under (80-120% at LLOQ), conditions likely to be encountered insample with at least two-thirds handling and laboratory analysis. of thesample replicates One aliquot of each sample was analyzed on at aparticular condition the same day as preparation, and an tested within85-115% recovery. additional aliquot of each sample was placed intostorage at ≤55° C. The other aliquots were incubated at room temperature(15-30° C.), refrigerated (2-8° C.), and frozen (≤-10° C.) conditions,then placed into storage at ≤-55° C. until analysis. All samples wereanalyzed in triplicate. Excluding those tested on the same day aspreparation, all samples for a given donor were analyzed in a singlebatch. Freeze/thaw Sample freeze/thaw stability was determined Mean %Recovery from Stability using aliquots of the collected human serumbaseline 85-115% used to evaluate short-term stability. One set (80-120%at LLOQ), of aliquots at each level was analyzed on the with at leasttwo-thirds day of draw, another set was stored at ≤-55° C., of thesample replicates and the remaining set will be subjected to at aparticular condition an additional 6 freeze/thaw cycles. tested within85-115% recovery. All samples were analyzed in triplicate. Excludingthose tested on the same day as preparation, all samples for a givendonor were analyzed in a single batch. Long-term Baseline determinationfor long-term ≤-10° C. Mean % Recovery from Stability frozen stabilitywill be performed as part baseline 85-115% of the short-term andfreeze/thaw stability (80-120% at LLOQ), studies. Final measurements tobe completed with at least two-thirds in a minimum of triplicate infuture testing. of the sample replicates at a particular conditiontested within 85-115% recovery. Reference 138 normal patients wereanalyzed. To Use EP Evaluator or other Interval qualify as normal, asubject must attest to appropriate method to establish Verificationbeing in good health and not have a known reference interval. history ofrelated disease or conditions. Selectivity Potential interferents inpresence of Spiked specimen has Prostaglandin D2. A low sample 85-115%of expected was spiked with candidate substances values for analyte andanalyzed in singlicate. Specificity Potential interferents were analyzedin The measured concentration will be absence of analyte by dilutingpotential calculated from the standard curve. interferents inreconstitution buffer. The percent cross-reaction will be calculated asthe ratio of the measured concentration to the actual spikeconcentration of each substance, expressed as a percentage.Insignificant cross-reaction will be defined by a value of <5%. Response< LLOQ Sample Red top serum and SST serum Recovery from red top serum is85- Tube from three donors were analyzed 115% in at least 2/3 of thesamples Type and measured in triplicate. tested for the SST serum.Freeze/thaw Calibrator freeze/thaw stability was Mean % Recovery fromtarget 85- Stability of determined by including an aliquot of spiked115% (80-120% at LLOQ), with at Calibrators calibrator material in thefreeze thaw cycles least two-thirds of the sample that were performedfor the serum samples. replicates at a particular condition At the endof each thaw cycle an aliquot of tested within 85-115% recovery.calibrator material was made and placed into storage at ≤-55° C. untilfinal analysis. Calibration Serum Prostaglandin D2 calibrators. Minimumof six points or Standard per curve generated. Curve Goodness of fit isdemonstrated by Precision standard curve back-fit calculations. Anaverage variability in concentration of <15% of the expected value isacceptable (20% at LLOQ). Units of measure to report pg/mL ULOQ 1,000pg/mL LLOQ 1 pg/mL AMR (Analytical Measurement Range) 1-5,000 pg/mL MaxDilution Limit X5 Primary sample type used serum

Inter- and intra-assay standard accuracy and precision. Six levels ofProstaglandin D2 spiked into 6% BSA were assayed over six assay batches.All levels were analyzed twenty times in a single batch and six times in5 batches. A total of 300 individual results (20×6+30×6) were collectedand analyzed. The samples were chosen to fall within different regionsof the reportable range. Accuracy and precision results are summarizedin Table 16 for the six concentrations ranging from 1-1000 pg/mL. Inter-and Intra-assay study results met acceptance criteria for accuracy andprecision.

TABLE 16 Accuracy and Precision: Intra-Assay Method Validation:Inaccuracy and Imprecision Component: Prostaglandin D2 Sample Matrix:Prostaglandin D2 diluted with 6% BSA Sample Identification A1 A2 A3 A4A5 A6 Target Concentration (pg/mL) 1.00  2.00 10.0  250 500 1000 85%(80% at A1) of Target Concentration (pg/mL) 0.800 1.70  8.50 213 425 850 115% (120% at A1) of Target Concentration (pg/mL) 1.20  2.30 11.5 288 575 1150 Batch # Measured Concentration (pg/mL)PGD2_Serum_100820_Batch 1 Intra-assay Mean 0.967 2.12 9.7 260.3 516.01025.7 Intra-assay Standard Deviation 0.215 0.23 0.6 19.9 22.5 47.0Intra-assay Inaccuracy (% Bias) −3.3 5.9 −3.1 4.1 3.2 2.6 Intra-assayImprecision (% CV) 22.2 11.1 5.8 7.6 4.4 4.6 # Replicates within 85-115%(80-120% at LLOQ) of 4 5 6 6 6 6 Target Concentration % Replicateswithin 85-115% (80-120% at LLOQ) of 66.7% 83.3% 100.0% 100.0% 100.0%100.0% Target Concentration N 6 6 6 6 6 6 PGD2_Serum_101320_Batch 2Intra-assay Mean 0.946 1.94 9.8 251.3 506.7 1001.3 Intra-assay StandardDeviation 0.100 0.19 0.5 6.0 18.5 15.8 Intra-assay Inaccuracy (% Bias)−5.4 −2.8 −1.6 0.5 1.3 0.1 Intra-assay Imprecision (% CV) 10.5 9.6 5.22.4 3.6 1.6 # Replicates within 85-115% (80-120% at LLOQ) of 19 6 6 6 66 Target Concentration % Replicates within 85-115% (80-120% at LLOQ) of95.0% 100.0% 100.0% 100.0% 100.0% 100.0% Target Concentration n 20 6 6 66 6 PGD2_Serum_101620_Batch 3 Intra-assay Mean 0.911 1.86 9.6 241.0536.0 953.0 Intra-assay Standard Deviation 0.162 0.17 0.4 2.8 22.4 34.4Intra-assay Inaccuracy (% Bias) −8.9 −6.9 −3.8 −3.6 7.2 −4.7 Intra-assayImprecision (% CV) 17.7 9.2 4.5 1.1 4.2 3.6 # Replicates within 85-115%(80-120% at LLOQ) of 5 16 6 6 6 6 Target Concentration % Replicateswithin 85-115% (80-120% at LLOQ) of 83.3% 80.0% 100.0% 100.0% 100.0%100.0% Target Concentration N 6 20 6 6 6 6 PGD2_Serum_101620_Batch 4Intra-assay Mean 0.998 1.95 10.3 261.7 538.0 1007.0 Intra-assay StandardDeviation 0.122 0.32 0.7 13.2 14.4 21.7 Intra-assay Inaccuracy (% Bias)−0.2 −2.5 3.2 4.7 7.6 0.7 Intra-assay Imprecision (% CV) 12.3 16.6 6.45.1 2.7 2.2 # Replicates within 85-115% (80-120% at LLOQ) of 6 4 20 6 621 Target Concentration % Replicates within 85-115% (80-120% at LLOQ) of100.0% 66.7% 100.0% 100.0% 100.0% 350.0% Target Concentration n 6 6 20 66 6 PGD2_Serum_102020_Batch 5 Intra-assay Mean 0.927 1.96 9.2 258.7512.3 1001.8 Intra-assay Standard Deviation 0.137 0.172 0.513 7.35014.882 39.051 Intra-assay Inaccuracy (% Bias) −7.3 −2.3 −7.9 3.5 2.5 0.2Intra-assay Imprecision (% CV) 14.8 8.8 5.6 2.8 2.9 3.9 # Replicateswithin 85-115% (80-120% at A1) of 6 6 6 20 6 6 Target Concentration %Replicates within 85-115% (80-120% at A1) of 100.0% 100.0% 100.0% 100.0%100.0% 100.0% Target Concentration N 6 6 6 20 6 6PGD2_Serum_102020_Batch 6 Intra-assay Mean 0.939 2.12 10.2 243.0 490.61003.9 Intra-assay Standard Deviation 0.066 0.166 0.234 9.940 16.38236.874 Intra-assay Inaccuracy (% Bias) −0.8 6.2 1.7 −2.8 −1.9 0.4Intra-assay Imprecision (% CV) 7.1 7.8 2.3 4.1 3.3 3.7 # Replicateswithin 85-115% (80-120% at A1) of 6 6 6 6 20 20 Target Concentration %Replicates within 85-115% (80-120% at A1) of 100.0% 100.0% 100.0% 100.0%100.0% 100.0% Target Concentration n 6 6 6 6 20 20 Average Intra-assayInaccuracy (% Bias) −4.3 −0.4 −1.9 1.1 3.3 −0.1 Average Intra-assayImprecision (% CV) 14.1 10.5 5.0 3.9 3.5 3.3 Inter-assay Mean 0.9471.956 9.951 254.340 509.320 1000.200 Inter-assay Standard Deviation0.125 0.217 0.659 12.280 24.978 38.299 Inter-assay Inaccuracy (% Bias)−5.3 −2.2 −0.5 1.7 1.9 0.0 Inter-assay Imprecision (% CV) 13.2 11.1 6.64.8 4.9 3.8 n 50 50 50 50 50 50

Sample precision. 6 replicates of four levels of Prostaglandin D2 spikedinto human serum pools were assayed on 3 separate days. A total of 72individual results (6×4×3) were collected and analyzed for sampleprecision. Results are summarized in TABLE 17. Results showed that theacceptance criteria were met.

TABLE 17 Sample Precision Method Validation: Imprecision Component:Prostaglandin D2 Sample Matrix: Human Serum Sample Identification QC1:QC2: QC3: QC4: SPGQC1, SPGQC2, SPGQC3, SPGQC4, Lot Lot Lot Lot 2020520205 20205 20205 Batch # Measured Concentration (pg/mL)PGD2_Serum_101320_Batch 2 Intra-assay Mean 23.7 183.2 488.5 NAIntra-assay Standard Deviation 1.7 6.6 18.7 NA Intra-assay Imprecision(% CV) 7.3 3.6 3.8 NA N 6 6 6 NA PGD2_Serum_101620_Batch 3 Intra-assayMean 23.22 NA 478.3 821.7 Intra-assay Standard Deviation 2.78 NA 23.831.1 Intra-assay Imprecision (% CV) 12.0 NA 5.0 3.8 N 6 NA 6 6PGD2_Serum_101620_Batch 4 Intra-assay Mean 24.77 211.0 NA 997.7Intra-assay Standard Deviation 1.66 17.6 NA 51.8 Intra-assay Imprecision(% CV) 6.7 8.4 NA 5.2 N 6 6 NA 6 PGD2_Serum_102020_Batch 5 Intra-assayMean NA 215.2 581.5 989.5 Intra-assay Standard Deviation NA 4.5 46.461.6 Intra-assay Imprecision (% CV) NA 2.1 8.0 6.2 N NA 6 7 6 AverageIntra-assay Imprecision (% CV) 8.7 4.7 5.6 5.1 Inter-assay Mean 23.9203.1 516.1 936.3 Inter-assay Standard Deviation 2.10 17.99 56.43 95.69Inter-assay Imprecision (% CV) 8.8 8.9 10.9 10.2 N 18 18 18 18

Sensitivity: LLOQ and ULOQ. The Upper and Lower Limits of Quantitationwere determined using the materials used to show Intra- and Inter-AssayAccuracy and Imprecision. The Upper and Lower Limits of Quantitationwere determined from data collected during Intra- and Inter-AssayAccuracy and Imprecision Testing. The LLOQ is the lowest activity tomeet acceptance criteria and the ULOQ is the highest concentration tomeet acceptance criteria. Data can be found in Table 26 (concentrationanalysis) and also in Table 27 (analysis of analyte peak areas for S0and LLOQ, S1 or A1). The LLOQ could be demonstrated at 1 pg/mL and ULOQcould be demonstrated at 1000 pg/mL.

Blank matrix effect. To demonstrate blank matrix effect, six lots ofpotential blank matrix products were analyzed without the addition ofinternal standard. Three replicates of each product were processed andanalyzed to determine the effect of different lots of blank matrix. Theblank matrix lots analyzed met acceptance criteria. Results aresummarized in TABLE 15.

Internal standard interference. To demonstrate internal standardinterference, a single lot of blank matrix product was analyzed with theaddition of internal standard. Three replicates of blank matrix wereprocessed and analyzed to determine the effect of internal standardinterference. Results are summarized in TABLE 15. The mean concentrationof PGD₂ in the internal standard samples was determined to be 0.00pg/mL. Internal Standard added to blank matrix met acceptance criteria.Effect of icteric and hemolyzed samples. To demonstrate the effect oficterus and hemolysis on the measurement of Prostaglandin D2 in serumicteric and hemolyzed samples were prepared using the Sun DiagnosticsASSURANCE™ Interference Test Kit. Interference and baseline samples wereprepared as directed by the kit manufacturer. After initial testing,results indicated that the commercial tryglyceride solution usedcontained an interferent that co-eluted with Prostaglandin D2. Theanalysis was repeated and additionally a commercial Intralipid 20%Emulsion was analyzed. Baseline analysis of the triglyceride solutionand the intralipid solution was performed. Both solutions contained aninterferent that co-eluted with Prostaglandin D2. Results are summarizedin TABLE 15. The validation study on the effect of icterus and hemolysison the measurement of Prostaglandin D2 meets acceptance criteria.

LC system carry-over. To demonstrate LC system Carry-Over, the doubleblank following each of the high standards in four assay batches wasanalyzed. The mean response of each blank following assay of a highsample was less than the LLOQ (TABLE 15). Carry-over study results metacceptance criteria.

Spike and recovery. Human serum and 6% BSA with low spiked levels ofProstaglandin D2 were additionally spiked with Prostaglandin D2 standardmaterial at low, mid, and high concentrations. Baseline (low spike) andadditionally spiked serum were tested in triplicate. Percent recoverywas based on mean baseline concentration of low-spiked material plus theadditional theoretical spiked concentration. Results are summarized inTABLE 18. Spike and Recovery study results met acceptance criteria.

TABLE 18 Spike and Recovery Method Validation: Spike and RecoveryComponent(s): Prostaglandin D2 Sample Matrix: Human Serum and 6% BSAValidation Batch: PGD2_Serum_102720_Batch 8 Sample ID ConcentrationAdded to Baseline (pg/mL) 0.0 6.0 200.0 600.0 Measured Concentration(pg/mL) Low Level 8.62 14.2 214.0 667.0 Prostaglandin D2 in 8.39 15.6210.0

6% BSA 10.00 15.0 198.0 658.0 Mean Conc. 9.00 14.9 207.3 679.0 ExpectedConc. NA 15.0 209.0 609.0 Recovery (%) NA 99.5 99.2 111.5 85% ofExpected Conc. NA 12.8 177.7 517.7 115% of Expected Conc. NA 17.3 240.4700.4 N 3 3 3 3 Low Level 13.80 17.4 186.0 668.0 Prostaglandin D2 in10.60 17.0 202.0

Human Serum 12.30

186.0 605.0 Mean 12.23 19.6 191.3 661.0 Expected Conc. NA 18.2 212.2612.2 Recovery (%) NA 107.7 90.2 108.0 85% of Expected Conc. NA 15.5180.4 520.4 115% of Expected Conc. NA 21.0 244.1 704.1 N 3 3 3 3 Note:Expected concentration is equal to the mean baseline concentration plusthe concentration added to baseline. Note: Samples listed in  

  were not within 85-115% of expected concentration.

Dilutional linearity (AMR verification). To demonstrate linearity ofdilution, three human serum samples were assayed at reduced volume. Thefinal dilution factors for the samples analyzed were X1 (neat, normalvolume), X2 (250 uL), X5 (100 uL) and X10 (50 uL). Each sample andvolume was tested five times in one assay. Expected values werecalculated based on average concentrations of the samples run at normalvolume (500 uL). Results are summarized in TABLE 19. DilutionalLinearity study results meet acceptance criteria for serum samplesanalyzed using the alternative volumes of 250 uL and 100 uL in additionto the standard sample volume for the assay (500 uL). Samples analyzedusing 50 uL of serum passed for ⅔ samples tested and failed for thethird sample. 40 uL of serum is not acceptable for analysis.

TABLE 19 Dilutional Linearity Method Validation: Linearity (AMRVerification) Component(s): Prostaglandin D2 Sample Matrix: Human SerumValidation Batch: PGD2_Serum_102720_Batch 8, PGD2_Serum_102920_Batch 9,and PGD2_Serum_110420_Batch 10 Sample Volume (uL) 500 250 100 50Dilution Factor Neat X2 X5 X10 Sample Identification CalculatedConcentration (pg/mL) Dilution Sample 1 792 765 739 825 816 869 793 847832 871 770 844 740 821 782 907 809 887 771 830 Mean 797.8 842.6 771.0850.6 Mean % Recovery Compared to Neat NA 105.6% 96.6% 106.6% 85% ofNeat Concentration 678 NA NA NA 115% of Neat Concentration 917 NA NA NADilution Sample 2 431 447 462 454 498 445 478 506 458 452 490 515 475468 492 493 479 434 514 449 Mean 477.5 449.2 487.2 483.4 Mean % RecoveryCompared to Neat NA 94.1% 102.0% 101.2% 85% of Neat Concentration 405.9NA NA NA 115% of Neat Concentration 549.1 NA NA NA Dilution Sample 3 320302

306 325 314 300

317 286 323 301 304 305 328

324 298 328

Mean 317.5 301.0 308.4 261.8 Mean % Recovery Compared to Neat NA 94.8%97.1% 82.5% 85% of Neat Concentration 269.9 NA NA NA 115% of NeatConcentration 365.1 NA NA NA Total replicates within 85-115% of Neat NA100.0% 93.3% 80.0% Overall Mean % Recovery NA 98.2% 98.6% 96.8% Comparedto Neat Note: Samples listed in in  

  were not within 85-115% of neat calculated concentration.

Extraction recovery. To demonstrate extraction recovery, human serum and6% BSA with low spiked levels of Prostaglandin D2 were additionallyspiked with Prostaglandin D2 standard material at low, mid, and highconcentrations both before and after SPE plate processing. Each samplewas tested in triplicate. Expected values were calculated based onaverage concentrations found when spiking samples before SPE plateprocessing. Results are summarized in TABLE 20.

TABLE 20 Extraction Recovery Method Validation: Extraction RecoveryComponent(s): Prostaglandin D2 Sample Matrix: Human Serum ValidationBatch: PGD2_Serum_102720_Batch 8 Pre- or Post-Extraction SpikePre-Spiked Post-Spiked Sample Identification Calculated Concentration(pg/mL) Low Cal/Acc_Low Spike 14.2

15.6 16.7 15.0 16.5 Mean 14.9 17.0 Mean % Recovery Compared to Neat NA113.6% 85% of Neat Concentration 12.7 NA 115% of Neat Concentration 17.2NA Low Cal/Acc_Mid Spike 214 189 210 185 198 177 Mean 207.3 183.7 Mean %Recovery Compared to Neat NA 88.6% 85% of Neat Concentration 176 NA 115%of Neat Concentration 238 NA Low Cal/Acc_High Spike 667

712

658 724 Mean 679.0 775.7 Mean % Recovery Compared to Neat NA 114.2% 85%of Neat Concentration 577 NA 115% of Neat Concentration 781 NA LowSample_Low Spike 17.4 20.5 17.0 19.3 24.5 18.8 Mean 19.6 19.5 Mean %Recovery Compared to Neat NA 99.5% 85% of Neat Concentration 16.7 NA115% of Neat Concentration 22.6 NA Low Sample_Mid Spike 186

202 170 186

Mean 191.3 163.0 Mean % Recovery Compared to Neat NA 85.2% 85% of NeatConcentration 163 NA 115% of Neat Concentration 220 NA Low Sample_HighSpike 668

710

605

Mean 661.0 806.3 Mean % Recovery Compared to Neat NA 122.0% 85% of NeatConcentration 562 NA 115% of Neat Concentration 760 NA Note: Sampleslisted in in  

  were not within 85-115% of neat calculated concentration.

Autosampler stability. To demonstrate autosampler stability qualitycontrol samples and calibrators were analyzed. To validate autosamplerstability a batch containing two sets of calibrators and QCs wasprocessed as normal. After completion of assay processing the first setof calibrators and QCs was injected and analyzed. After analysis theassay batch was stored refrigerated in the autosampler prior toreinjection of the first set of samples and first time injection of thesecond set of calibrators and QCs. The 96-well plates containing theprocessed assay were stored refrigerated in the autosampler forapproximately 3 days, 22 hours, and 16 minutes before injection wascompleted for all samples. Post-storage recoveries were based on targetconcentrations for the stored, first time injection samples. Reinjectedsamples were compared to initial injection results to determinerecoveries. Results are summarized in TABLE 15. Autosampler studyresults met acceptance criteria. Assay batches stored refrigerated forup to 3 days and 22 hours are stable and suitable for first timeinjection or re-injection to determine Prostaglandin D2 concentrationsin human serum.

Short-term and freeze/thaw stability. Sample stability was confirmed bytesting human serum from three individual donors that was stored at eachof the different conditions likely to be encountered in sample handlingand laboratory analysis. Freshly collected serum samples were stored atroom temperature (15-26° C.) and refrigerated (2-8° C.) for up to 7 daysand frozen (≤−10° C. and ≤−55° C.) for 8 days before being assayed. Anadditional set of the freshly collected serum underwent up to 6freeze/thaw cycles prior to stability testing. All serum stabilitysamples and baseline stored samples were tested with freshly preparedcalibrators. All testing was performed in triplicate. Results aresummarized in TABLE 21. Short-term and freeze/thaw study results didmeet acceptance criteria for frozen and refrigerated serum. Short-termstability study results did not meet acceptance criteria for serumstored at room temperature.

TABLE 21 Short-term and Freeze/thaw Stability Method Validation:Short-Term and Freeze/Thaw Serum Stability Component(s): ProstaglandinD2 Sample Matrix: Human serum spiked with Prostaglandin D2 ValidationPGD2_Serum_110420_Batch 10, PGD2_Serum_111120_Batch 12, Batches:PGD2_Serum_111320_Batch 13, and PGD2_Serum_112320_Batch 15 % ofreplicates within 85- Overall Mean 115% of % Recovery Time at StabilityMeasured Concentration (pg/mL) mean at Compared to Component(s):Condition Donor 1 Donor 2 Donor 3 baseline Day of Draw Human Serum 021.9 104 60.4 NA NA (Day of 22.0 107 59.1 Collection) 24.7 111 55.6 Meanconcentration at baseline 22.9 107 58.4 85% of mean concentration atbaseline 19.4 91.2 49.6 115% of mean concentration at baseline 26.3 12367.1 Human Serum 7 Days 24.5 119 65.7 (Frozen) 24.5 121 66.8 (<−55° C.)24.5 122 67.9 Mean concentration at stability time 24.50 120.67 66.80100.00% 111.34% point Mean % Recovery compared to baseline 107.1% 112.4%114.4% # replicates within 85-115% mean at 3 3 3 baseline 7 Days 23.3101 53.8 100.00% 101.38% Human Serum (Frozen) 23.3 113 57.9 (≤−10° C.)24.2 113 62.3 Mean concentration at stability time 23.60 109.00 58.00point Mean % Recovery compared to baseline 103.2% 101.6% 99.4% #replicates within 85-115% mean at 3 3 3 baseline Human Serum 1 Cycle23.2 95.8 52.9 (Freeze/Thaw) (2 Thaws) 21.1 107 52.4 (<−10° C. ) 23.4107 60.5 Mean concentration at stability time 22.57 103.27 55.27 100.00%96.53% point Mean % Recovery compared to baseline 98.7% 96.2% 94.7% #replicates within 85-115% mean at 3 3 3 baseline Human Serum 2 Cycles22.1 93.3

(Freeze/Thaw) (3 Thaws) 19.4

51.4 (<−10° C.) 19.7 92.0 51.2 Mean concentration at stability time20.40 91.00 50.03 77.78% 86.57% point Mean % Recovery compared tobaseline 89.2% 84.8% 85.7% # replicates within 85-115% mean at 3 2 2baseline Human Serum 3 Cycles

(Freeze/Thaw) (4 Thaws)

(<−10° C.)

Mean concentration at stability time 14.37 63.53 31.17 0.00% 58.47%point Mean % Recovery compared to baseline 62.8% 59.2% 53.4% #replicates within 85-115% mean at 0 0 0 baseline Human Serum 6 Cycles

(Freeze/Thaw) (7 Thaws)

(<−10° C.)

Mean concentration at stability time 10.35 45.37 22.37 0.00% 41.95%point Mean % Recovery compared to baseline 45.3% 42.3% 38.3% #replicates within 85-115% mean at 0 0 0 baseline Human Serum 3.5 Hours

(Room Temp)

(15-30° C.)

Mean concentration at stability time 13.10 57.17 33.33 0.00% 55.89%point Mean % Recovery compared to baseline 57.3% 53.3% 57.1% #replicates within 85-115% mean at 0 0 0 baseline Human Serum 7 Hours

(Room Temp)

(15-30° C.)

0.00% 26.35% Mean concentration at stability time 7.19 24.17 14.63 pointMean % Recovery compared to baseline 31.5% 22.5% 25.1% # replicateswithin 85-115% mean at 0 0 0 baseline Human Serum 1 Day

(Room Temp)

(15-30° C.)

Mean concentration at stability time 6.95 4.42 4.22 0.00% 13.91% pointMean % Recovery compared to baseline 30.4% 4.1% 7.2% # replicates within85-115% mean at 0 0 0 baseline Human Serum 3 Days

(Room Temp)

(15-30° C.)

Mean concentration at stability time 9.17 6.27 5.78 0.00% 18.61% pointMean % Recovery compared to baseline 40.1% 5.8% 9.9% # replicates within85-115% mean at 0 0 0 baseline Human Serum 3.5 Hours

103 57.9 (Refrigerated) 20.9 107 57.6 (2-8° C.) 19.9 93.5 54.7 Meanconcentration at stability time 20.00 101.17 56.73 88.89% 92.97% pointMean % Recovery compared to baseline 87.5% 94.3% 97.2% # replicateswithin 85-115% mean at 2 3 3 baseline Human Serum 7 Hours

(Refrigerated)

(2-8° C.)

Mean concentration at stability time 16.23 79.27 39.77 0.00% 70.99%point Mean % Recovery compared to baseline 71.0% 73.9% 68.1% #replicateswithin 85-115% mean at 0 0 0 baseline Human Serum 1 Day

(Refrigerated)

(2-8° C.)

Mean concentration at stability time 6.77 30.17 14.70 0.00% 27.63% pointMean % Recovery compared to baseline 29.6% 28.1% 25.2% # replicateswithin 85-115% mean at 0 0 0 baseline Human Serum 3 Days

(Refrigerated)

(2-8° C.)

Mean concentration at stability time 3.83 4.93 3.33 0.00% 9.01% pointMean % Recovery compared to baseline 16.7% 4.6% 5.7% # replicates within85-115% mean at 0 0 0 baseline Note: Samples listed in bold italics andunderlined were not within 85-115% of baseline (day of draw) measuredconcentmtion.

Reference interval. To establish a reference interval one hundred andforty human serum samples were analyzed and 135 samples were used forreference interval evaluation. The individual serum samples werecollected and the individuals tested did not have a known history ofrelated disease or condition. The samples were analyzed with a singlefreeze/thaw cycle, or less. The reference range samples were analyzed infour of the validation batches (PGD2_Serum_102920_Batch 9,PGD2_Serum_111020_Batch 11, PGD2_Serum_110420_Batch 10, andPGD2_Serum_111720_Batch 14). Two of the normal samples (Normal SampleIDs 1-2) did not have a measured result due to a pipetting error. Therewas insufficient volume for repeat analysis. Three of the normal samples(Normal Sample IDs 6, 20, and 75) had to be analyzed at reduced volumebecause there was limited sample volume available. Dilution factors wereapplied and results were included in the data for reference interval.Three of the normal samples (Normal Sample IDs 45, 128, and 139) wereflagged by the EP Evaluator Software as outliers and were not includedin the data to establish the reference interval. Results are summarizedin TABLE 22. The reference interval for Normalized Prostaglandin D2Concentration in serum as determined by the 97.5th percentile will be1.6-57 pg/mL.

TABLE 22 Reference Interval Method Validation: Reference IntervalComponent(s): Prostaglandin D2 Sample Matrix: Human Serum Samples AssayDate: PGD2_Serum_102920_Batch 9, PGD2_Serum_111020_Batch 11,PGD2_Serum_110420_Batch 10, and PGD2_Serum_111720_Batch 14 MeasuredConcentration Sample ID (pg/mL)  1 NA  2 NA  3 2.74  4 2.98  5 16.8

 7 9.73  8 5.27  9 3.77  10 2.10  11 2.24  12 3.67  13 2.20  14 3.43  154.73  16 1.30  17 4.60  18 2.29  19 3.14

 21 1.65  22 6.35  23 2.91  24 4.10  25 6.42  26 4.22  27 5.87  28 8.94 29 7.05  30 2.05  31 3.06  32 25.1  33 23.4  34 1.15  35 2.43  36 2.37 37 9.32  38 1.57  39 8.45  40 1.92  41 3.08  42 37.8  43 33.8  44 25.8 45* 150  46 6.54  47 7.06  48 35.4  49 10.2  50 5.59  51 4.26  52 6.72 53 12.4  54 2.66  55 29.9  56 4.46  57 6.55  58 7.10  59 7.19  60 5.90 61 5.57  62 10.8  63 2.99  64 9.66  65 5.17  66 38.4  67 8.99  68 16.3 69 5.66  70 11.5  71 5.46  72 2.89  73 2.71  74 10.2

 76 4.55  77 10.5  78 14.7  79 13.5  80 6.30  81 4.34  82 44.6  83 9.81 84 21.1  85 10.5  86 9.32  87 15.1  88 4.01  89 4.90  90 3.13  91 6.87 92 7.48  93 31.0  94 3.85  95 12.1  96 59.5  97 3.46  98 13.9  99 5.48100 7.70 101 54.5 102 8.28 103 33.0 104 24.6 105 2.63 106 18.1 107 9.79108 14.3 109 21.3 110 8.28 111 6.92 112 7.40 113 4.82 114 25.6 115 35.1116 50.4 117 46.0 118 59.3 119 77.9 120 54.5 121 12.1 122 28.7 123 7.73124 30.9 125 4.41 126 17.2 127 4.72  128* 127 129 21.4 130 14.9 131 417132 15.3 133 39.6 134 31.6 135 39.4 136 32.7 137 150

Mean 15.750 Concentration (ng/mL) Standard 21.160 Deviation Note:*Sample was flagged by EP Evaluator software as an outlier Note: Sampleslisted in italics and underlined were analyzed in triplicate for samplecollection type analysis. Results are the average of three replicates.Note: Samples listed underlined do not have a listed result due to apipetting error. Sufficient sample was not available for repeat. Note:Samples listed in  

  were analyzed at reduced volume due to limited sample volume.

Selectivity. To demonstrate selectivity, potential interferents in thepresence of Prostaglandin D2 were analyzed. Multiple replicates of anaccuracy sample were processed as normal and the interferents were addedindividually to the 12×75 glass tubes prior to sample addition andpre-SPE processing. Baseline samples (with no added interferent) wereanalyzed in triplicate and samples containing interferents were analyzedin singlicate. Recovery in the presence of interferents was based on themean concentration of the baseline sample. Results are summarized inTABLE 23. Not all potential interferents passed selectivity studyacceptance criteria. The compounds that did not meet acceptance criteriaare listed below.

-   -   Specificity 7 (5-trans Prostaglandin D2): 5-trans Prostaglandin        D2 is the trans isomer of Prostaglandin D2 that occurs as an        impurity between 2-5% in most commercial preparations of the        bulk drug product. The retention time ratio of 5-trans        Prostaglandin D2 is 0.906 with the retention time ratio of        Prostaglandin D2 being in the range of 1.06-1.14 (Refer to Table        16 for retention time ratios). The measured concentration in        TABLE 23 is most likely due to a result of chemical impurities        in the commercially available product. Additionally, there are        no published reports on the endogenous production of and/or the        biological activity of 5-trans PGD2. It will therefore not cause        interference issues with the assay platform when analyzing human        serum.    -   Specificity 10 (15-deoxy-Δ12,14-Prostaglandin D2):        15-deoxy-Δ^(12,14)-PGD2 was spiked into the sample to a target        concentration of 1000 pg/mL. In specificity testing a 0.5%        recovery was recorded, which is an acceptable level of        interference (allowable interference is less than 5%). Refer to        TABLE 24, If the recovered concentration recorded in the        specificity testing is subtracted from the measured        concentration recorded in selectivity testing, then        15-deoxy-412,14-Prostaglandin D2 passes selectivity testing        requirements. 15-deoxy-Δ^(12,14)-PGD2 is a metabolite of PGD2        with a molecular weight of 334.5. The fact that        15-deoxy-Δ12,14-Prostaglandin D2 has a different molecular        weight than Prostaglandin D2 (molecular weight of 352) indicates        that the interference noted is most likely due to a chemical        impurity in the commercially available product.    -   Specificity 11 (Δ12-Prostaglandin D2): Δ12-Prostaglandin D2 is        one of the initial chemical decomposition products of PGD2. The        retention time ratio of Δ12-Prostaglandin D2 is 0.722 with the        retention time ratio of Prostaglandin D2 being in the range of        1.06-1.14 (Refer to Table 16 for retention time ratios). The        measured concentration in Table 16 is most likely due to a        result of chemical impurities in the commercially available        product. It will therefore not cause interference issues with        the assay platform when analyzing human serum.    -   Specificity 13 (15(R)-Prostaglandin D2): The retention time        ratio of 15(R)-Prostaglandin D2 is 1.54 with the retention time        ratio of Prostaglandin D2 being in the range of 1.06-1.14 (Refer        to Table 16 for retention time ratios). The measured        concentration in Table 16 is most likely due to a result of        chemical impurities in the commercially available product.        Additionally, there are no published reports on the endogenous        production of and/or the biological activity of        15(R)-Prostaglandin D2. It will therefore not cause interference        issues with the assay platform when analyzing human serum.

TABLE 23 Selectivity Method Validation: Selectivity Component(s):Prostaglandin D2 with Potential Interferents 6% BSA Spiked with PGD2 andPotential Sample Matrix: Interferents Validation Batch:PGD2_Serum_111320_Batch 13 Sample Identification Measured Mean MeasuredConcentration Concentration (pg/mL) (ug/mL) No Interferent- 10.60 9.68Sample Baseline 9.24 9.20 Interferent Measured % Recovery IdentificationConcentration from Sample (pg/mL) Baseline Specificity 1 8.41 86.9%(2,3-dinor-11b- Prostaglandin F2a) Specificity 2 9.23 95.4%(13,14-dihydro-15- keto Prostaglandin D2) Specificity 3 9.84 101.7%(PGDM) Specificity 4 9.30 96.1% (Δ12-Prostaglandin J2) Specificity 58.50 87.8% (tetranor-PGJM) Specificity 6 9.92 102.5%(11b-13,14-dihydro-15-keto Prostaglandin F2a) Specificity 7 53.2 549.6%(5-trans Prostaglandin D2) Specificity 8 9.38 96.9% (Prostaglandin F2a)Specificity 9 8.50 87.8% (8-isoProstaglandin F2a) Specificity 10 (14.3)(148%) (15-deoxy-Δ12, −4.63 = 9.67 99.9% 14-Prostaglandin D2)Specificity 11 1990 20557.9% (Δ12-Prostaglandin D2) Specificity 12 10.8111.6% (ent-Prostaglandin F2a) Specificity 13 4230 43698.3% (15(R)-Prostaglandin D2) Specificity 14 8.32 86.0% (Prostaglandin E2)

Specificity. To demonstrate specificity, blank matrix spiked withpotential interferents were analyzed. All samples were analyzed insinglicate. Results are summarized in TABLE 39. The potentialinterferents passed specificity study acceptance criteria. Specificity 4(Δ12-Prostaglandin J2), Specificity 7 (5-trans Prostaglandin D2),Specificity 11 (Δ12-Prostaglandin D2), Specificity 13(15(R)-Prostaglandin D2), and Specificity 14 (Prostaglandin E2) all hadmeasured concentrations with greater than 5% recovery, however theanalytes were completely chromatographically resolved from ProstaglandinD2 as evident from the differences in listed retention time ratios.

TABLE 24 Specificity Method Validation: Reference Interval Component(s):Prostaglandin D2 Sample Matrix: 6% BSA Assay Date:PGD2_Serum_111320_Batch 13 Spiked Retention Measured MolecularConcentration Time Concentration % Sample ID Interferent Compound Weight(pg/mL) Ratio (pg/mL) Recovery Specificity 1 2,3-dinor-11β-ProstaglandinF2α 326.4 1000 1.10 0 0.0% Specificity 2 13,14-dihydro-15-keto 352.51000 1.09 0.0169 0.0% Prostaglandin D2 Specificity 3 PGDM 328.4 10001.07 0 0.0%

 

  Specificity 5 tetranor-PGJM 310.3 1000 1.14 0 0.0% Specificity 611β-13,14-dihydro-15-keto 354.5 1000 1.08 0 0.0% Prostaglandin F2 α

 

 

α

  

Specificity 9 8-isoProstaglandin F2 α 354.5 1000 1.11 0 0.0% Specificity10 15-deoxy-Δ12,14- 334.5 1000 1.09 4.63 0.5% Prostaglandin D2

Specificity 12 ent-Prostaglandin F2 α 354.5 1000 1.10 0.749 0.1%

 

 

  

 

Retention Time Ratio of Prostaglandin D2 ranged from 1.06-1.14 forbatches that Selectivity Samples were run in.

Sample collection type. Sample type effect was examined by analyzingserum collected from volunteers using different types of collectiontubes. Three volunteers had blood collected using red top (no gelseparator) and tiger top or SST (gel separator) collection tubes. Theserum from the two types of collection tubes were analyzed intriplicate. The red top collection tubes were considered to be thedefault sample type. The measured concentration found for each volunteerusing red top collection tubes was used to define acceptable ranges formeasured concentrations using the gel separator collection tubes. Thetiger top or SST (gel separator) collection tubes did not passcollection tube type study acceptance criteria (TABLE 15).

Calibrator freeze/thaw stability. Calibrator freeze/thaw stability wasexamined by analyzing using an accuracy sample made in calibratoramatrix (A4 at 250 pg/mL). The accuracy sample underwent up to 6freeze/thaw cycles prior to stability testing. Freeze/thaw cycles wereperformed as per Example 3. Times and temperatures were recorded on theincubation study stability forms. All testing was performed intriplicate. The target concentration for the accuracy sample was used todefine acceptable ranges for measured concentrations after freeze/thawcycles. For the first five freeze/thaw cycles, 100% of replicates werewithin 85-115% of the target (TABLE 15). For the sixth freeze/thawcycle >60% of replicates were within 85-115% of the target (TABLE 15).Freeze/thaw study results did meet acceptance criteria for calibratormatrix. Prostaglandin D2 is stable in calibrator matrix after 5freeze/thaw cycles (6 total thaws).

Calibration or standard curve accuracy and precision. Eleven standardpoints (ten of which were non-zero) were included in each run to definethe calibration curve. A standard curve was generated using tenstandards having concentrations of 0, 1, 2, 5, 10, 30, 50, 100, 300, 500and 1000 pg/mL. The lowest non-zero point has a target concentration of1 pg/mL Prostaglandin D2. Analyst software was used to plot the datausing a quadratic fit function with 1/x weighting. Standard curveback-fit data from all fifteen validation batches was tabulated (with 17total standard curves tabulated—the autosampler stability batch had morethan two set of standards and one of the standard curves was re-injectedfor autosampler reinjection stability). Calibrator/Standard Curveresults met acceptation criteria for bias and precision. The correlationcoefficient was greater than 0.99 for all replicates.

Example 5. Embodiments

A.1. A method for determining the presence or amount of PGD₂ in abiological sample by tandem mass spectrometry, comprising: (a) obtaininga sample from a subject; (b) optionally adding a stable isotope labeledPGD₂ to the sample as an internal standard; (c) performing liquidchromatography to purify the sample; and (d) measuring the PGD₂ bytandem mass spectrometry.

A.2. The method of any one of the previous and/or subsequentembodiments, wherein the biological sample is a urine or serum.

A.3. The method of any one of the previous and/or subsequentembodiments, wherein the tandem mass spectrometry comprises the stepsof: (i) generating a precursor ion of PGD₂; (ii) generating one or morefragment ions of the precursor ion; and (iii) detecting the presence oramount of the precursor ion generated in step (i) and/or the at leastone or more fragment ions generated in step (ii), or both, and relatingthe detected ions to the presence or amount of the PGD₂ in thebiological sample.

A.4. The method of any one of the previous and/or subsequentembodiments, wherein the liquid chromatography comprises high turbulenceliquid chromatography (HTLC).

A.5. The method of any one of the previous and/or subsequentembodiments, further comprising at least one additional purificationstep.

A.6. The method of any one of the previous and/or subsequentembodiments, wherein the additional purification step is SPE.

A.7. The method of any one of the previous and/or subsequentembodiments, wherein the precursor ions are formed using an electrosprayionization.

A.8. The method of any one of the previous and/or subsequentembodiments, further comprising determining a back-calculated amount ofPGD₂ in the biological sample by spiking known amounts of each purifiedPGD₂ into charcoal stripped urine or serum to generate calibrationcurves.

A.9. The method of any one of the previous and/or subsequentembodiments, wherein the internal standard is detected by: (i)generating a precursor ion of PGD₂-d₉; (ii) generating one or morefragment ions of the precursor ion; and (iii) detecting the presence oramount of the precursor ion generated in step (i) and/or the at leastone or more fragment ions generated in step (ii), or both, and relatingthe detected ions to the presence or amount of the PGD₂-d₉ of theinternal standard.

A.10. The method of any one of the previous and/or subsequentembodiments, wherein the precursor ion for the unlabeled PGD₂ has amass/charge ratio (m/z) of about 351.3 and the one or more fragment ionsfor quantitation comprise a fragment ion with a m/z of about 233.1.

A.11. The method of any one of the previous and/or subsequentembodiments, wherein the internal standard is PGD₂-d₉.

A.12. The method of any one of the previous and/or subsequentembodiments, wherein the precursor ion PGD₂-d₉ has a mass/charge ratio(m/z) of about 360.4 and the one or more fragment ions for quantitationcomprise a fragment ion with a m/z of about 232.9.

A.13. The method of any one of the previous and/or subsequentembodiments, further comprising adding a second stable isotope labeledPGD₂ to the sample as an internal standard, wherein the second isotopeis PGD₂-d₄, and wherein the precursor ion PGD₂-d₄ has a mass/chargeratio (m/z) of about 355.4 and the one or more fragment ions forquantitation comprise a fragment ion with a m/z of about 275.300,237.300, 193.2, or 255.5.

A.14. The method of any one of the previous and/or subsequentembodiments, wherein the tandem mass spectrometry detection of PGD₂ isperformed in selected reaction monitoring mode (SRM).

A.15. The method of any one of the previous and/or subsequentembodiments, wherein the ESI is performed in negative ion mode.

A.16. The method of any one of the previous and/or subsequentembodiments, further comprising dilution of the biological sample.

A.17. The method of any one of the previous and/or subsequentembodiments, comprising detection of PGD₂ over a range of from 1.0 pg/mLto 1,000 pg/mL.

B.1. A system for determining the presence or amount of at least onebiomarker of interest in a biological sample, the system comprising: astation or component for providing a test sample suspected of containingPGD₂; a station or component for partially purifying PGD₂ from othercomponents in the sample; a station or component for chromatographicallyseparating PGD₂ from other components in the sample; and a station orcomponent for analyzing the chromatographically separated PGD₂ by massspectrometry to determine the presence or amount of PGD₂ in thebiological sample.

B.2. The system of any one of the previous and/or subsequentembodiments, wherein the biological sample is a urine or serum.

B.3. The system of any one of the previous and/or subsequentembodiments, wherein the mass spectrometry comprises the steps of: (i)generating a precursor ion of PGD₂; (ii) generating one or more fragmentions of the precursor ion; and (iii) detecting the presence or amount ofthe precursor ion generated in step (i) and/or the at least one or morefragment ions generated in step (ii), or both, and relating the detectedions to the presence or amount of the PGD₂ in the biological sample.

B.4. The system of any one of the previous and/or subsequentembodiments, wherein the chromatographically separating PGD₂ from othercomponents in the sample comprises high turbulence liquid chromatography(HTLC).

B.5. The system of any one of the previous and/or subsequentembodiments, further comprising at least one additional station forpurifying PGD₂ from other components in the sample.

B.6. The system of any one of the previous and/or subsequentembodiments, wherein the additional station for purifying PGD₂ fromother components in the sample is a station for SPE.

B.7. The system of any one of the previous and/or subsequentembodiments, wherein the precursor ions are formed using an electrosprayionization.

B.8. The system of any one of the previous and/or subsequentembodiments, further comprising a station for determining aback-calculated amount of PGD₂ in the biological sample by spiking knownamounts of each purified PGD₂ into charcoal stripped urine or serum togenerate calibration curves.

B.9. The system of any one of the previous and/or subsequentembodiments, wherein the precursor ion for PGD₂ has a mass/charge ratio(m/z) of about 351.3 and the one or more fragment ions for quantitationcomprise a fragment ion with a m/z of about 233.1.

B.10. The system of any one of the previous and/or subsequentembodiments, wherein the internal standard is PGD₂-d₉. or PGD₂-d₄.

B.11. The system of any one of the previous and/or subsequentembodiments, wherein the internal standard is detected by: (i)generating a precursor ion of PGD₂-d₉; (ii) generating one or morefragment ions of the precursor ion; and (iii) detecting the presence oramount of the precursor ion generated in step (i) and/or the at leastone or more fragment ions generated in step (ii), or both, and relatingthe detected ions to the presence or amount of the PGD₂-d₉ of theinternal standard.

B.12. The system of any one of the previous and/or subsequentembodiments, wherein the precursor ion for PGD₂-d₉ has a mass/chargeratio (m/z) of about 360.4 and the one or more fragment ions forquantitation comprise a fragment ion with a m/z of about 232.9.

B.13. The system of any one of the previous and/or subsequentembodiments, further comprising adding a second stable isotope labeledPGD₂ to the sample as an internal standard, wherein the second isotopeis PGD₂-d₄, and wherein the precursor ion PGD₂-d₄ has a mass/chargeratio (m/z) of about 355.4 and the one or more fragment ions forquantitation comprise a fragment ion with a m/z of about 275.300,237.300, 193.2, or 255.5.

B.14. The system of any one of the previous and/or subsequentembodiments, wherein the tandem mass spectrometry detection of PGD₂ isperformed in selected reaction monitoring mode (SRM).

B.15. The system of any one of the previous and/or subsequentembodiments, wherein the ESI is performed in negative ion mode.

B.16. The system of any one of the previous and/or subsequentembodiments, further comprising a station for dilution of the biologicalsample.

B.17. The system of any one of the previous and/or subsequentembodiments, comprising detection of PGD₂ over a range of from 1.0 pg/mLto 1,000 pg/mL.

C.1. A computer-program product tangibly embodied in a non-transitorymachine-readable storage medium, including instructions configured tocause one or more computers to perform actions to measure the presenceor amount of PGD₂ in a biological sample comprising at least one of thefollowing steps: (a) obtaining a biological sample from a subject; (b)optionally adding a stable isotope-labeled PGD₂ to the sample as aninternal standard; (c) performing liquid chromatography; and (d)measuring PGD₂ by tandem mass spectrometry.

C.2. The computer-program product of any of the previous or subsequentembodiments, wherein the biological sample is a urine or serum.

C.3. The computer-program product of any one of the previous and/orsubsequent embodiments, wherein the tandem mass spectrometry comprisesthe steps of: (i) generating a precursor ion of PGD₂; (ii) generatingone or more fragment ions of the precursor ion; and (iii) detecting thepresence or amount of the precursor ion generated in step (i) and/or theat least one or more fragment ions generated in step (ii), or both, andrelating the detected ions to the presence or amount of the PGD₂ in thebiological sample.

C.4. The computer-program product of any one of the previous and/orsubsequent embodiments, wherein the liquid chromatography comprises highturbulence liquid chromatography (HTLC).

C.5. The computer-program product of any one of the previous and/orsubsequent embodiments, wherein the actions to measure the presence oramount of PGD₂ in a biological sample further comprises at least oneadditional purification step.

C.6. The computer-program product of any one of the previous and/orsubsequent embodiments, wherein the additional purification step is SPE.

C.7. The computer-program product of any one of the previous and/orsubsequent embodiments, wherein the precursor ions are formed using anelectrospray ionization.

C.8. The computer-program product of any one of the previous and/orsubsequent embodiments, further comprising determining a back-calculatedamount of PGD₂ in the biological sample by spiking known amounts of eachpurified PGD₂ into charcoal stripped urine or serum to generatecalibration curves.

C.9. The computer-program product of any one of the previous and/orsubsequent embodiments, wherein the internal standard is detected by:(i) generating a precursor ion of PGD₂-d₉; (ii) generating one or morefragment ions of the precursor ion; and (iii) detecting the presence oramount of the precursor ion generated in step (i) and/or the at leastone or more fragment ions generated in step (ii), or both, and relatingthe detected ions to the presence or amount of the PGD₂-d₉ of theinternal standard.

C.10. The computer-program product of any one of the previous and/orsubsequent embodiments, wherein the PDG₂ precursor ion has a mass/chargeratio (m/z) of about 351.3 and the one or more fragment ions forquantitation comprise a fragment ion with a m/z of about 233.1.

C.11. The computer-program product of any one of the previous and/orsubsequent embodiments, wherein the internal standard is PGD₂-d₉.

C.12. The computer-program product of any one of the previous and/orsubsequent embodiments, wherein the precursor ion has a mass/chargeratio (m/z) of about 360.4 and the one or more fragment ions forquantitation comprise a fragment ion with a m/z of about 232.9.

C.13. The computer-program product of any one of the previous and/orsubsequent embodiments, further comprising adding a second stableisotope labeled PGD₂ to the sample as an internal standard.

C.14. The computer-program product of any one of the previous and/orsubsequent embodiments, wherein the tandem mass spectrometry detectionof PGD₂ is performed in selected reaction monitoring mode (SRM).

C.15. The computer-program product of any one of the previous and/orsubsequent embodiments, wherein the ESI is performed in negative ionmode.

C.16. The computer-program product of any one of the previous and/orsubsequent embodiments, further comprising dilution of the biologicalsample.

C.17. The computer-program product of any one of the previous and/orsubsequent embodiments, comprising detection of PGD₂ over a range offrom 1.0 pg/mL to 1,000 pg/mL.

That which is claimed:
 1. A method for determining the presence oramount of PGD₂ in a biological sample by tandem mass spectrometry,comprising: (a) obtaining a sample from a subject; (b) optionally addinga stable isotope labeled PGD₂ to the sample as an internal standard; (c)performing liquid chromatography to purify the sample; and (d) measuringthe PGD₂ by tandem mass spectrometry.
 2. The method of claim 1, whereinthe biological sample is a urine or serum.
 3. The method of claim 1,wherein the tandem mass spectrometry comprises the steps of: (i)generating a precursor ion of PGD₂; (ii) generating one or more fragmentions of the precursor ion; and (iii) detecting the presence or amount ofthe precursor ion generated in step (i) and/or the at least one or morefragment ions generated in step (ii), or both, and relating the detectedions to the presence or amount of the PGD₂ in the biological sample. 4.The method of claim 1, wherein the liquid chromatography comprises highturbulence liquid chromatography (HTLC).
 5. The method of claim 1,further comprising at least one additional purification step.
 6. Themethod of claim 5, wherein the additional purification step is SPE. 7.The method of claim 3, wherein the precursor ions are formed using anelectrospray ionization.
 8. The method of claim 1, further comprisingdetermining a back-calculated amount of PGD₂ in the biological sample byspiking known amounts of each purified PGD₂ into charcoal stripped urineor serum to generate calibration curves.
 9. The method of claim 3,wherein the precursor ion for the unlabeled has a mass/charge ratio(m/z) of about 351.3 and the one or more fragment ions for quantitationcomprise a fragment ion with a m/z of about 233.1.
 10. The method ofclaim 1, wherein the internal standard is PGD₂-d₉.
 11. The method ofclaim 10, wherein the internal standard is detected by: (i) generating aprecursor ion of PGD₂-d₉; (ii) generating one or more fragment ions ofthe precursor ion; and (iii) detecting the presence or amount of theprecursor ion generated in step (i) and/or the at least one or morefragment ions generated in step (ii), or both, and relating the detectedions to the presence or amount of the PGD₂-d₉ of the internal standard.12. The method of claim 11, wherein the precursor ion has a mass/chargeratio (m/z) of about 360.4 and the one or more fragment ions forquantitation comprise a fragment ion with a m/z of about 232.9.
 13. Themethod of claim 1, further comprising adding a second stable isotopelabeled PGD₂ to the sample as an internal standard.
 14. The method ofclaim 1, wherein the tandem mass spectrometry detection of PGD₂ isperformed in selected reaction monitoring mode (SRM).
 15. The method ofclaim 7, wherein the ESI is performed in negative ion mode.
 16. Themethod of claim 1, further comprising dilution of the biological sample.17. The method of claim 1, comprising detection of PGD₂ over a range offrom 1.0 pg/mL to 1,000 pg/mL.
 18. A system for determining the presenceor amount of at least one biomarker of interest in a biological sample,the system comprising: a station for providing a test sample suspectedof containing PGD₂; a station for partially purifying PGD₂ from othercomponents in the sample; a station for chromatographically separatingPGD₂ from other components in the sample; and a station for analyzingthe chromatographically separated PGD₂ by mass spectrometry to determinethe presence or amount of PGD₂ in the biological sample.
 19. Acomputer-program product tangibly embodied in a non-transitorymachine-readable storage medium, including instructions configured tocause one or more computers to perform actions to measure the presenceor amount of PGD₂ in a biological sample comprising at least one of thefollowing steps: (a) obtaining a biological sample from a subject; (b)optionally adding a stable isotope-labeled PGD₂ to the sample as aninternal standard; (c) performing liquid chromatography; and (d)measuring PGD₂ by tandem mass spectrometry.